CFSE-MLR and cell sorting protocol to define alloreactive T cell repertoires by high throughput TCR sequencing

Human 6-day CFSE MLR PROTOCOL

 Sykes Lab

MLR MEDIA:

          For 500 ml MLR Media, sterile filter the following:

                    470 ml AIMV

                    25ml human serum* [final: 5%]

                    5 ml HEPES stock (1M, 1:100) [final:10mM] 

                    1 ml 2ME stock (25mM, 1:500) [final: 50uM]

*Human serum is heat inactivated (56°C, 30mins) to destroy complement activity.

Centrifuge condition if not otherwise specified: 4 °C, 450g, 5mins

DAY 0  Set-Up: Assay must be done using sterile technique in tissue culture hood

1. Prepare Human whole PBMCs or cells form other tissues (spleen, lymph nodes): wash twice in MLR media after thawing. Re-suspend in MLR media and count. Note that sorting T cells as responders will only detect directly alloreactive clones.

1. Alternative if perform cell labeling with CFSE or Violet Proliferation Dye right away: After the first wash in MLR media to get rid of DMSO freezing media, use PBS to reconstitute cell pellets to an appropriate volume for counting, after counting, spin cells down, which is considering as PBS wash.
2. Given downstream labeling need to avoid serum, will use PBS to resuspend cells and split in half for two-way (GvH, HvG) MLR (if applicable).

2. Stain Stimulators with Violet Proliferation Dye 450 (VPD450: BD Horizon; store in aliquots (5ul/tube) at -80 °C)

1. Spin and re-suspend stimulator cells in PBS 10-30 million/ ml (make sure there is no excess MLR media upon re-suspension as that will reduce the brightness of the stain)
2. Add 1 μL violet dye per ml of cells
3. Vortex and incubate at 37 degrees for 10-15 minutes (12 minutes)
4. Add 9x the volume of PBS to wash; spin
5. Re-suspend in MLR; spin
6. Re-suspend in MLR at concentration of 2 million/ml

3. Irradiate stimulators (35 gray)

Irradiator: X-RAD 320;

Manuel mode:

height: 40cm

35Gy=525 Seconds

KV: 320

mA: 12.5

filter: 1

4. Stain responders with CFSE

a. Spin and re-suspend responders in PBS at 1 million/ml (15 ml or 50 ml tube)

b. Make CFSE dye

    A. FROM WHOLE VIAL (50ug; M.W.: 557.47 g/M):

1. Take 18 μl DMSO and transfer into CFSE individual stock tube (5uM/ml)
2. Take 10 μl of this mixture and add to 990 μl of PBS (1:100), vortex
3. Stain with 10 μl CFSE/ml cells (some use 4μl/ml)

    B. FROM DILUTED CFSE (1mM solution in DMSO) [50ug CFSE /90ul DMSO]

1. use 0.5ul/ml cells

c. Vortex and leave at room temperature for 2 minutes, in the dark

d. Quench with serum or MLR medium and put on ice for 5 minutes

e. Wash 2-3 times with MLR at 4°C

f. Re-suspend at final concentration of 2 million/ml in MLR

5. Plate cells

a. Plate cells in a 96 well plate, round-bottom

1. Per well: 100 μl responder (200,000) + 100 μl stimulator (200,000)
2. Do not plate in outside border rows of cells
3. Can also use 24 well plate with 1 million responders and 1 million stims

b. Plate 1-2 wells with just CFSE-stained responders for compensation control and responder only control

c. Plate 1-2 wells with just Violet-stained stimulators for compensation control and stimulator only control

d. Other conditions to consider plating

1. Responder against self (prepare according to steps 2&3 above)
2. Responder against media
3. Responder with PHA

e. Consider plating unstained cells for compensation control (if applicable) upon harvesting、

6. Place 200 μl PBS in outside wells

7. Seal plate with Scotch Brand Magic Tape (Step 7 & 8 helps in reducing evaporation and prevents contamination).

8. Incubate plates at 37 °C, 5% CO₂ until day 6.

DAY 6: Harvest

9. Harvest cells into sterile FACS tubes or 15/50 ml conical tubes, count cells and adjust live cells to 10 million/ml for staining

10. Stain cells with desired antibodies (e.g., CD45, CD3, gd TCR, CD4, CD8)

 a. Keep in mind that CFSE is in FITC channel and VPD is in the same channel as Pac Blue and DAPI for our BD Influx sorter.

 b. Example staining: CD45 V500, CD3 PerCP-Cy5.5, gdTCR PE-Cy7, CD4 AF700, CD8 APC-Cy7, DAPI

1. Adjust antibody/FACS buffer volumes so that there is 10ul antibody per million cells with a total volume of 100ul per million cells

11. CELL SORTING

a. Cells must be in polypropylene tubes for sorting (not in polystyrene tube)

b. Cells must be filtered IMMEDIATELY before sorting

c. Cells can be sorted into up to 4 polypropylene tubes. Always put MLR media (0.5-1 ml) in the bottom of the tubes you are sorting into

d. Gating strategy:

1. Include lymphocyte blasts in forward-scatter/side-scatter
2. Gate VPD450/DAPI- CD45+ CD3+ responders
3. After gating on CD3+ gdTCR- cells, show CD4 vs CD8, and gate on the CFSE low CD4 and CD8 ab T cells, pushing the gate close to the CFSE high population while still feeling confident that all CFSE low cells have divided at least once. Alternatively, to better avoid sorting overlap with undivided cells, can move sort gate further away from the undivided population.

12. DNA Extraction

a. Use QIAGEN Blood & Tissue DNA extraction kit

    a. Transfer post-sorting cells from polypropylene tubes to 15ml conical tubes for PBS wash to have well-formed cell pellet

    b. Wash cells once with PBS before DNA extraction

    c. Optional: If cell number is less than 100,000, can use 1ul RNA carrier (stored in -20 °C)

    d. Elute cells in 30-50 ul of AE buffer, depending on cell number and downstream TCR-seq pipeline requirements

    e. Store DNA samples at -20 °C

For TCR repertoire analysis, please refer to our published article:

Obradovic A, Shen Y, Sykes M, Fu J. Integrated analysis toolset for defining and tracking alloreactive T-cell clones after human solid organ and hematopoietic stem cell transplantation. Software Impacts. 2021 Nov 1;10:100142. 3