In vitro Anti-tumor TIL expansion experiment

In vitro Anti-tumor TIL expansion experiment

Sri Krishna and Frank J Lowery, Surgery Branch, NCI, 01/08/2021

Goal: To identify anti-tumor ecognition and total cell yield upon repeated expansions from different TIL subsets.

1. Thaw tumor infiltrating T cells (TILs) from infusion product and rest overnight in a T25 flask in 10mL of TIL-media without any cytokines
2. TIL subsets from the infusion product (CD8+ or CD39- CD69- or CD39+ CD69+) were isolated (5e5 total cells per subset) by flow cytometry-based cell sorting. 

Note: Sort for more than 5e5 cells to account for sample loss.

1. Rest flow sorted TIL subsets for an hour without cytokines
2. Harvest and count autologous patient target tumor cell line
3. Set up tumor-TIL co culture by co-incubating 1e5 TIL subset with 1e5 tumor cells per well in a 96 well plate in 100uL TIL-media without cytokines overnight (5 wells each for each condition).
4. Next day, use 41BB (CD137) based sorting to isolate tumor-reactive flow-sorted TIL subset (41BB+). 
5. 1e5 tumor-reactive 41BB+ cells are sorted for each TIL-subset that has been cocultured (e.g. CD8+ 41BB+ or CD8+ CD39- CD69- 41BB+ or CD8+ CD39+ CD69+ 41BB+). 

Note: Sort for more than 1e5 cells to account for sample loss. 

1. Perform rapid expansion protocol (REP): each 1e5 sorted 41BB+ TIL subsets with 2e7 irradiated allogeneic feeders (50 Gy), 30ng/mL OKT3 antibody (anti-CD3), and 3000 U IL-2 in a T25 flask (day 0) in TIL-media.

Note: Have a T25 control flask with irradiated feeders alone to ensure feeders do not outgrow.

1. Check for expansion every 2-3 days. On day 5 if activated cell clusters are apparent initiate media change using 6000U IL-2. Note: Do not add OKT3 from now on.
2. Continue media change and TIL expansion into T75 or bigger flasks if needed for two weeks from starting REP. 
3. Count cells and set up autologous tumor coculture as in step #5. Readout can be done on IFNg ELISpot or 41BB upregulation overnight. Or cells can be frozen to be assayed for tumor-recognition later.
4. Same day, set up second round of re-expansion if needed using 1e5 TILs from REP1 and 2e7 irradiated allogeneic feeders as shown in step #8. 
5.  Follow steps 8-11, if needed. 
6. If subsequent TIL REPs are needed to be assayed for anti-tumor recognition, freeze all samples down and perform anti-tumor TIL recognition together. 
7. Count total cell yield at each step from each REP. Calculate the theoretical cell yield from normalizing to total cell yield from each REP step. 

Antibodies:

Anti-CD8-PE-Cy7 (RPA-T8, 1:300, BD Biosciences, USA)

Anti-CD39-FITC (A1; 1:200, BioLegend, USA)

Anti-CD69-APC (FN50; 1:25, BD Biosciences, USA)

Anti-CD137-PE (4B4-1,  BD Bisociences, USA)

Category