TITLE: TCR sequencing and activation
AUTHORS: Zorea Jonathan

[DESCRIPTION]
This protocol is a procedure we follow to isolate tumor infiltrating T cells and try to activate them with unique peptides and to characterize their TCR repertoire 

[STEPS]
SECTION: Tumor implantation
1. Grow sufficient amount of cells (5*10^6 per mouse). At the day cells are ready to be inject, thaw a vial of Basement Membrane Extract (Biotest, 3433-010-01) on ice. Keep 200ul tips in -20° as well.
2. Once the BME is thawed, trypsinize the cells, count them using trypan blue, and resuspend them in PBS in the concentration of 5M cells in 40ul PBS. add 10ul of BME per 40ul of cells suspension and keep pn ice. 
** BME should be always kept on ice 

SECTION: Mice treatment
5. A day before treatment initiation, suspend GDC-0077 in MCT buffer to reach a dose of 25mg/kg.


 
 
MCT buffer recipe 

 


Keep The drug on a magnetic stirrer over night. 
3. Inject 50ul of cells solution to the lip of each mouse using 300ul syringe. 
4. Allow cells to grow until tumor size reach the size of 4X4 (5-8 days)
SECTION: Mice treatment
6. The next day, gavage each mice with 200ul of the drug solution daily. 
Measure tumor size using the caliper at the first day of treatment and every 3-4 days. 
SECTION: Tumor + lymph node collection
8. Arrange enough plain RPMI medium and ice before start sacrificing mice!
SECTION: Tumor + lymph node collection
9. Sacrifice the mice, cage by cage, and collect tumors and mandibular lymph bodes using scissors and forceps. Keep the correct attribution for each set of tumor + lymph nodes. Place everything on ice in the RPMI medium.
SECTION: Mice treatment
7. Keep treating the mice for 7-10 days, until the effect of the drug can be seen. 
SECTION: Single cell isolation
10. Tumors:  Chop 1-2 tumors on a 60mm petri plate using scalpel, until all big chunks are gone. 
SECTION: Single cell isolation
10.1. Collect all the content of the plate using plain RPMI. Once you finished chopping all of the tumors, centrifuge all the tubes, waste the supernatant, and resuspend the tumors in 5ml of an enzymatic dissociation cocktail:
 


SECTION: Single cell isolation
10.2. Transfer all the content to a gentleMACS™ C Tubes, place the C tubes on the gentleMACS machine, and run an appropriate protocol 
 

SECTION: Single cell isolation
10.3. Transfer all the C tube content to a 15 or 50ml tube, centrifuge for 5min on 300g, and waste the supernatant. Resuspend the tumor cells in 4ml RPMI.
SECTION: Single cell isolation
11. Lymphs: Place a 70um strainer on top of a 50ml tube. Place the lymph nodes on top of the strainer together with RPMI. Using the "leg" of a 3ml syringe, press the lymph nodes over the mesh. Collect all the single cells by washing the mesh with RPMI. 
SECTION: Single cell isolation
11.1. Centrifuge for 5min on 300g, and collect the pellet. 
SECTION: Single cell isolation
12. Resuspend the cells pellet with 3ml of sterile AKC buffer for 3 min.
Add 9ml of PBS and centrifuge for 5min at 300g. Aspirate the supernatant and resuspend the cells in T-cells medium. Count the cells.

T-cells medium
Table 3. T cell culture mediumComponentFinal concentrationAmountRPMI 1640 with L-glutaminen/a445 mLPenicillin-streptomycin solution (10×)1×5 mL2-mercaptoethanol (1 M)50 μM25 μLFBS10% (v/v)50 mL
 

ACK Lysis Buffer
   ReagentQuantity (for 1000 mL)Final concentrationNH4Cl 8.02 g150 mMKHCO3   1 g  10 mMNa2EDTA 37.2 mg 0.1 mM

 
 
SECTION: Single cell isolation
10.4. Insert a glass pipet to the bottom of the 15ml tube, blocking air tranfer with your thumb. With a syringe, add 2ml of Ficoll-Paque PLUS, making sure not to mix it.
SECTION: Single cell isolation
10.5. Centrifuge the tubes for 10min, 350g with no brakes. 
SECTION: Single cell isolation
10.6. Collect only the lymphocytes phase to a new 15 ml tube and wash it with PBS. Aspirate the supernatant and resuspend the cells in PBS.
Count the cells and resuspend up to 10^7 cells in 40ul MACS buffer. (same amount for each sample)
Keep the pellet of CD8+ isolation.

MACS buffer
0.5% bovine serum albumin (BSA)
2 mM EDTA
in PBS
SECTION: CD8+ isolation
13. Following the CD8a+T Cell Isolation Kit mouse Order no. 130-104-075 protocol of Miltenyi :

Add 10 µL of Biotin-Antibody Cocktail per 10⁷ total cells.
SECTION: CD8+ isolation
14. Mix well and incubate for 5 minutes in the refrigerator (2−8 °C).
SECTION: CD8+ isolation
15. Add 30 µL of buffer per 10⁷ total cells.
SECTION: CD8+ isolation
16. Add 20 µL of Anti-Biotin MicroBeads per 10⁷ total cells.
SECTION: CD8+ isolation
17. Mix well and incubate for 10 minutes in the refrigerator (2−8 °C).
SECTION: CD8+ isolation
18. Proceed to subsequent magnetic cell separation. 
▲ Note: A minimum of 500 µL is required for magnetic separation. If necessary, add buffer to the cell suspension.
SECTION: CD8+ isolation
19. Place LS Column in the magnetic field of a suitable MACS Separator. For details refer to the respective MACS Column data sheet.
SECTION: CD8+ isolation
20. Prepare column by rinsing with 3 mL of buffer.
SECTION: CD8+ isolation
21. Apply cell suspension onto the column. Collect flow-through containing unlabeled cells, representing the enriched CD8a+ T cells.
SECTION: CD8+ isolation
22. Wash column with 3 mL of buffer. Collect unlabeled cells that pass through, representing the enriched CD8a+ T cells, and combine with the effluent from step 21.
SECTION: Analyze positive population by Flow
25. Fc-block cells with 25ul TruStain fcX™ (anti-mouse CD16/32) fc blocker(1:1000) diluted in FACS buffer for 5-10 minutes on ice.

FACS buffer
1% FCS
1mM EDTA
0.05% Na-Azid
in PBS
SECTION: CD8+ isolation
23. Remove column from the separator and place it on a suitable collection tube. Pipette 5 mL of buffer onto the column. Immediately flush out the magnetically labeled nonCD8a+ T cells by firmly pushing the plunger into the column.
SECTION: CD8+ isolation
24. Centrifuge both labeled and unlabeled cells 5min at 300g, and resuspend both in 1ml of T-cell medium. Take 50ul of each for the Flow cytometry validation. 
SECTION: Analyze positive population by Flow
26. Add 25ul of appropriately conjugated fluorescent primary antibodies at predetermined optimum concentrations (1:300 by default) and incubate on ice for 15-20 minutes in the dark.

** dilute antibodies in FACS buffer. It is recommended to create a stock solution of each antibody following the next calculation:
2ul of AB stock will be diluted in a total of 25ul, so stock should be 12.5X. 300/12.5 = 24. If we take 1ul of an antibody and dilute it with 23ul of FACS buffer, we then create a stock we can use 2ul of it per sample. This will allow us to mix up to 12 antibodies and no exceeding the 25ul.    
 
SECTION: Analyze positive population by Flow
27. prepare DAPI solution by diluting DAPI in FACS buffer to a concentration of 3ug/ml, and add 100ul of this solution per sample.   
SECTION: Analyze positive population by Flow
28. Analyze the positive and negative population over the Flow cytometer.
SECTION: CD8+ T cells RNA isolation
29. Spin cells for 5 min at 300 X g. Remove media and resuspend cells in ice cold PBS. Pellet cells by spinning at 300 X g for 5 min. Lyse cells with TRIZOL Reagent by repetitive pipetting or by passing through syringe and needle. Use 1 ml of the reagent per 5-10 X 10^6 of animal cells.
SECTION: CD8+ T cells RNA isolation
30. Incubate the homogenized sample for 5 minutes at room temperature to permit the complete dissociation of nucleoprotein complexes. Centrifuge to remove cell debris. Transfer the supernatant to new tube.
SECTION: CD8+ T cells RNA isolation
31. Add 0.2 ml of chloroform per 1 ml of TRIZOL Reagent. Cap sample tubes securely.
Vortex samples vigorously for 15 seconds and incubate them at room temperature for
2 to 3 minutes.
SECTION: CD8+ T cells RNA isolation
32. Centrifuge the samples at no more than 12,000 x g for 15 minutes at 2
to 80C. 
 

SECTION: CD8+ T cells RNA isolation
33. Transfer upper aqueous phase carefully without disturbing the interphase into fresh tube. Measure the volume of the aqueous phase (The volume of the aqueous phase is about 60% of the volume of TRIZOL Reagent used for homogenization).
SECTION: CD8+ T cells RNA isolation
34. Precipitate the RNA from the aqueous phase by mixing with isopropyl alcohol. Use 0.5
ml of isopropyl alcohol per 1 ml of TRIZOL Reagent used for the initial homogenization.
Incubate samples at 15 to 30oC for 10 minutes and centrifuge at not more than 12,000
x g for 10 minutes at 2 to 4oC. The RNA precipitate, often invisible before
centrifugation, forms a gel-like pellet on the side and bottom of the tube. 
SECTION: CD8+ T cells RNA isolation
35. Remove the supernatant completely. Wash the RNA pellet once with 75% ethanol,
adding at least 1 ml of 75% ethanol per 1 ml of TRIZOL Reagent used for the initial
homogenization. Mix the samples by vortexing and centrifuge at no more than 7,500 x
g for 5 minutes at 2 to 8 oC. Repeat above washing procedure once. Remove all
leftover ethanol. 
SECTION: CD8+ T cells RNA isolation
36. Air-dry or vacuum dry RNA pellet for 5-10 minutes. Do not dry the RNA pellet by
centrifuge under vacuum. It is important not to let the RNA pellet dry completely as
this will greatly decrease its solubility. Partially dissolved RNA samples have an A260/A280
ratio < 1.6. Dissolve RNA in DEPC-treated water by passing solution a few times
through a pipette tip.
SECTION: CFSE staining of lymph cells
37. Prepare a 2 µM working solution by diluting 1 µL of 5 mM CFSE stock solution in 2.5 mL PBS
SECTION: CFSE staining of lymph cells
38. Spin down and resuspend cells at 10-100 x 10^6 cells/mL in the CFSE working solution.
SECTION: CFSE staining of lymph cells
39. Incubate cells for 20 minutes at room temperature and keep protected from light.
SECTION: CFSE staining of lymph cells
40. Quench the staining by adding 5 times the original staining volume of cell culture medium containing 10% FBS.
SECTION: CFSE staining of lymph cells
41. Pellet cells and resuspend in pre-warmed cell culture medium.
SECTION: CFSE staining of lymph cells
42. Count the cells and dilute them to 200k/100ul in T-cells medium. 
SECTION: Incubate peptides with lymph node cells
43. The 24 peptides are in a stock concentration of 2mM. Dilute the peptides 1:100 in T-cells medium and place them according to the map below :
 

SECTION: Incubate peptides with lymph node cells
44. Seed the cells from step 42 in the 24 wells of the peptide + 2 No peptides well + PMA+ION well as a positive control.
SECTION: Incubate peptides with lymph node cells
45. Allow cells to grow 48-72 hours. Quantify CFSE together with CD8+ using FACS.