Analysis of Human T Cell Activity in an Allogeneic Co-Culture Setting of Pre-Treated Tumor Cells

Tumor cells change the immune phenotype during therapy, which might impact on the outcome and therefore the aim of our research is to investigate the interaction between T cells and tumor cells to create a better understanding of those interactions.

Enhancing radio sensitivity, particularly in radio-resistant tumors by combining radiotherapy to kinase inhibitors. Targeting the DNA damage response is currently a central part of recent developments. Three-dimensional cell culture models and co-culture models are currently established in the field of radiobiology to increase the comparability between in vitro and in vivo data.

[Narrator] To begin, obtain HSC4 tumor cells in a suitable volume of D10 medium. Seed at least three wells per condition, each containing 15,000 cells in 200 microliters of medium in 96 well plates. Designate one plate for sample treatment, including wells for tumor cell only and T-cell only control and wells for cell counting for each treatment on the co-culture day. On day two, treat the tumor cells with kinase inhibitors at the required concentrations. Irradiate one plate after three to four hours and the same after 24 hours of incubation with five gray for hypofractionated irradiation. For peripheral blood mononuclear cells, or PBMC isolation, transfer the EDTA mixed blood collected from a healthy donor into two 50 milliliter centrifugation tubes. Fill both centrifugation tubes up to 50 milliliters with PBS and 2% FBS. Next, prepare six centrifugation tubes with plastic inlays for PBMC separation. Fill each with 15 milliliters of cold density gradient medium. Carefully overlay the density gradient media with 12 to 15 milliliters of the diluted blood. Centrifuge the tubes at 1,200 G for 10 minutes without deceleration. After centrifugation, transfer the supernatant into four new 50 milliliter centrifugation tubes. Discard the used tubes. Fill the new centrifugation tubes up to 50 milliliters with PBS and 2% FBS and centrifuge at 120 G for 10 minutes at room temperature. Carefully discard the supernatant. Resuspend the cell pellet in one milliliter of PBS containing 2% FBS and combine the two pellets into one falcon tube. Fill the centrifugation tube to 50 milliliters again with PBS containing 2% FBS. Centrifuge the PBMCs at 300 G for 10 minutes. Carefully discard the supernatant. Resuspend the cell pellet in 80 microliters of MACS plus buffer per 10 to the power of seven cells. Add 20 microliters of CD8 microbeads per 10 to the power of seven cells and carefully mix by pipetting up and down. Incubate for 15 minutes at four degrees celsius. After incubation, wash the cells with two milliliters of MACS plus buffer per 10 to the power of seven cells. Centrifuge the tube at 300 G for 10 minutes at room temperature. After discarding the supernatant, resuspend the cells in 1000 microliters of MACS plus buffer. Set two MS columns in the magnet and place two 15 milliliter centrifugation tubes underneath. Equilibrate the columns with 500 microliters of MACS plus buffer. Then load an equal volume of the cell suspension into the prepared MS column. Rinse the columns with 500 microliters of MACS plus buffer three times. Label the final collected cells as flow through or CD8 negative and combine them in one single centrifugation tube. Then take a new 15 milliliter centrifugation tube labeled CD8 positive T cells. Flush both columns containing magnetically labeled CD8 positive T cells with 1000 microliters of MACS plus buffer each. Before staining the T-cells, centrifuge them at 300 G for five minutes. Discard the supernatant and resuspend the cell pellet in 1000 microliters of PBS. Centrifuge the suspension again at 300 G for five minutes. After discarding the supernatant, resuspend the cell pellet in 2000 microliters of one micromolar CFSE staining solution. Incubate for 20 minutes at 37 degrees Celsius. Next, centrifuge the stained cells at 300 G for five minutes. After washing the cells with PBS, resuspend them in T-cell medium. Seed the cells into the CD3 and CD28 coated six well plate and incubate. On day four, harvest the T cells and resuspend them in their respective medium. Add the desired number of T-cells to the tumor cell wells in a ratio of one to one after counting the designated wells. On day seven, remove the culture plate from the incubator for quantification of T cell proliferation. After washing the cells, stain them with the desired antibodies and perform flow cytometry to assess the T cells. Exclude the doublet population based on the forward scatter area versus forward scatter height to obtain singlets. Plot the singlets to identify T cells based on the forward scatter area versus the side scatter area. Plot the CD3 expression against the side scatter area to discriminate T cells. Further, identify CD8 positive T cells after plotting the CD8 signal against the side scatter area. All T cells should express CD3 and CD8. Plot the CFSE signal of all CD8 positive T cells as a histogram. After completing the gating steps, select CD8 positive T cells as input and analyze their CD25 or HLA-DR isotype expression by plotting the respective surface marker fluorescence against the side scatter area. The proportion of proliferative T cells increased when co-cultured with irradiated HSC4 tumor cells. Radiation therapy or RT and RT plus inhibition of ATR significantly enhanced T cell proliferation compared to RT plus ATM inhibition. Among the highly proliferative T cells, RT plus ATR inhibition was the most effective in stimulating proliferation. Expression of CD25 on T cells was downregulated after co-culture with RT-treated HSC4 tumor cells. Pre-treatment of tumor cells with ATM inhibitor alone significantly decreased CD25 expression compared to ATR inhibitor. The combination of RT and ATM inhibitor resulted in increased CD25 expression compared to RT plus ATR inhibitor. HLA-DR expression on T cells was generally upregulated by RT. Co-culture with HSC4 cells pretreated with RT plus ATR inhibitor further increased HLA-DR expression compared to RT alone or RT plus ATM inhibitor.