Real Time Detection of In Vitro Tumor Cell Apoptosis Induced by CD8⁺ T Cells to Study Immune Suppressive Functions of Tumor-infiltrating Myeloid Cells

Our protocol assesses the effects of myeloid-derived suppressor cells and the tumor-associated macrophages in T-cell-induced tumor cell apoptosis. Alongside investigation of the underlying mechanisms of potential therapeutic interventions. This protocol directly evaluates T-cell-induced tumor cell apoptosis with high sensitivity and enables longitudinal analysis and imaging of cell-to-cell interactions.

The efficacy of checkpoint inhibitor is best looked at by tumor-infiltrating myeloid cells in certain tumor types. This technique could lead to the identification of novel therapies for these tumors. This method could not only advance cancer immunology research, but could potentially also provide insight into the immunodeficiency and autoimmune diseases.

For the activation and expansion of spleen-isolated CD8-positive T-cells, first aliquot one times 10 to the fifth CD8-positive T-cells in 50 microliters of E-DMEM into individual wells of a 96-well U-bottom plate. Next, add one times 10 to the fifth suppressor cells in 50 microliters of E-DMEM, or 50 microliters of E-DMEM alone, into each well of T-cells. Then add 50 microliters of freshly-prepared activation medium and 50 microliters of E-DMEM, with or without the test reagents of interest, to the appropriate wells.

And place the plate in a 37-degree-Celsius and 5%carbon dioxide incubator with 95%humidity for four days. To set up a pre-activated CD8-positive T-cell target cell co-culture, first add 30 microliters of 1-to-100-diluted growth-factor-reduced soluble basement membrane matrix to the appropriate wells of a 96-well flat-bottom plate, suitable for microscopy. Shake the plate to spread the matrix evenly, and place the plate in the cell culture incubator for at least one hour.

While the plate is equilibrating, prepare the target cells. Aspirate the medium, wash with PBS, and add one milliliter of 05%Trypsin-EDTA at room temperature for one minute. Add nine milliliters of DMEM supplemented with fetal bovine serum by gentle pipetting, and transfer the dissociated cells into tubes.

After centrifuging the cells, resuspend the pallet in 500 microliters of E-DMEM. Filter the resuspended cells through a cell strainer, and count the live cells. Then adjust the density to four times 10 to the fourth target cells per milliliter in fresh E-DMEM on ice.

Next, pipette the pre-activated CD8-positive T-cells a few times to resuspend the cells into a single cell suspension, and transfer the floating T-cells into one 1.5-milliliter tube per condition. Collect any remaining cells with 200 microliters of PBS per well, transfer the washes into the appropriate 1.5-milliliter tubes, and centrifuge. Aspirate the medium and add one milliliter of E-DMEM to each tube for centrifugation.

After centrifugation, resuspend the pellets in 100 microliters of fresh E-DMEM per tube. After counting, adjust the cells in each tube to a density of 1.6 times 10 to the fifth pre-activated CD8-positive T-cells per milliliter of medium, and place the cells on ice. When the cells are ready, aspirate the basement membrane matrix from each well of the 96-well microscopy plate, and add 50 microliters of target cells to individual wells within the inner 60 wells of the plate with mixing.

Add 25 microliters of E-DMEM supplemented with four times 10 to the third units per milliliter of IL2, and 10-micromolar fluorogenic caspase-3 substrate to the appropriate wells. Then add 25 microliters of pre-activated CD8-positive T-cells to the appropriate wells. And finally, add E-DMEM to the appropriate wells to make a total volume of up to 100 microliters.

Add 200 microliters of PBS or sterile water into all of the empty wells. Shake the plate, and leave the plate on a flat surface for 10 minutes. To image the cells, set the microscope to acquire images in phase contrast.

As well as fluorescence channels suitable for the nuclear-restricted fluorescent protein, and the fluorogenic activated caspase-3 substrate fluorophores used in the experiment. Then capture images of each experimental well in phase contrast and the two fluorescence channels every one to three hours for at least 72 hours. To analyze the captured images, open an image from a control well containing only target cells and medium without caspase-3 substrate in the fluorescence channel used for substrate signal.

Observe whether an inappropriate fluorescence signal is being emitted by the nuclei. If the substrate signal is apparent within the nuclei, use spectral unmixing to increase the percentage of substrate signal removed from the nuclei signal, until the substrate signal disappears. Next, view a control well containing only nuclei unlabeled effector cells in medium without caspase-3 substrate in the two fluorescence channels individually.

Observe whether a signal is emitted by the nuclei. If no signal is apparent in the individual channels, no spectral unmixing is necessary. To resolve the fluorescent objects in both fluorescence channels, use a fluorescence background subtraction method with relevant parameters for the sample and the appropriate parameters for edge splitting.

Use images of target cell monoculture to establish parameters for target cell nuclear fluorescence. Use images of effector cell monoculture to establish parameters for substrate-induced apoptotic nuclear fluorescence. Use images of co-culture to verify or refine parameters for substrate-induced apoptotic nuclear fluorescence in target cells.

To determine the minimum size of the target nuclei, use images in the nuclei signal channel from the wells containing only target cells with caspase substrate. To determine the average size of apoptotic effector nuclei, use images in the substrate signal channel from the wells containing only effector cells with caspase substrate. To count the number of fluorescence target cell nuclei, set up an analysis procedure using an appropriate minimum size restriction.

Using effector cell monoculture images, set up a second analysis procedure to count the number of apoptotic nuclei that are larger than the mean size of the apoptotic effector nuclei. Then set up a third analysis procedure to count the number of apoptotic target cells by counting the number of nuclei in which the nucleus signal and size-restricted substrate signal significantly co-localize. Typically, cancer cells increase the fluorescence signal in their nuclei following the activation of a nuclear-targeting caspase biosensor when the cells make contact with CD8-positive T-cells pre-activated by antibodies in the absence of suppressor cells.

The nucleus sizes of CD8-positive T-cells are smaller than those of cancer cells. Thus, apoptotic effector cells can be excluded from apoptotic target cell counts by a size restriction image analysis method. Although some target cancer cells exhibit a small rounded shape without substrate fluorescence, this does not affect the analysis.

As these cells are undergoing mitosis rather than apoptosis and thus are excluded from apoptotic target cell counts by a nuclear substrate signal overlap mask. The co-culture of target cancer cells with pre-activated CD8-positive T-cells increases tumor cell apoptosis above the levels of spontaneous apoptosis in cancer cell monocultures. Generally, when using an optimal ratio of target cancer cells to effector cells, a peak in the number of apoptotic target cancer cells can be observed.

This peak becomes more distinct when the data is expressed as the apoptotic fraction of the target cell population. The most important thing to remember when attempting this protocol is to set up monocultures of target cells and monocultures of effector cells for use in the developing the analysis masks. This method can provide insight into the duration and frequency of T-cell to cancer cell interactions, and the conditions associated with gene-dependent cytotoxicity in both human and mouse cells.

We are currently developing this assay into a high-throughput screen using human cells to identify compounds that inhibit myeloid cell-mediated immunosuppression and thereby enhance checkpoint inhibitor efficacy.