TITLE: Mitochondrial antigen presentation (MitAP) to primary 2C CD8 T (proliferation and suppression) assay
AUTHORS: Alexandra Kazanova, Samantha Gruenheid

[DESCRIPTION]
This protocol details the mitochondrial antigen presentation (MitAP) to primary 2C CD8 T (proliferation and suppression) assay. In order to assess MitAP in an assay that incorporated all signals for naïve T cells activation in vivo, we tested whether H.pylori-exposed BMDCs could trigger activation of mitochondria-reactive primary CD8 T cell.

[STEPS]
SECTION: Bone Marrow-derived Dendritic cells (BMDC, 0-9 days of experiment):
1. Femurs from three Pink1+/+ and three Pink1-/- mice are cleaned manually from soft tissues and flushed with ice cold PBS.
SECTION: Bone Marrow-derived Dendritic cells (BMDC, 0-9 days of experiment):
2. Cells are pelleted and 2 mL of ACK RBC lysis buffer are added for 1 min and mixed gently with 1 ml pipette tip.

SECTION: Bone Marrow-derived Dendritic cells (BMDC, 0-9 days of experiment):
3. Lysis buffer is blocked with 13 mL of complete RPMI and pelleted with centrifugation.

SECTION: Bone Marrow-derived Dendritic cells (BMDC, 0-9 days of experiment):
4. Cells are resuspended, counted with hemocytometer and brough to 200,000 cell/ml in complete RPMI.
SECTION: Bone Marrow-derived Dendritic cells (BMDC, 0-9 days of experiment):
5. 10 ng/ml of mr-GMCSF is added to BMDC for differentiation.

SECTION: Bone Marrow-derived Dendritic cells (BMDC, 0-9 days of experiment):
6. Cells are plated in non-tissue culture treated plasticware (6 well plate, sarstedt) for 4320 min at 37 °C.

SECTION: Bone Marrow-derived Dendritic cells (BMDC, 0-9 days of experiment):
7. On day 3 equal volume of the starting culture of complete RPMI with 10 ng/ml mr-GMCS is gently added on top of the well (avoid any additional shaking of the cells).
SECTION: Bone Marrow-derived Dendritic cells (BMDC, 0-9 days of experiment):
8. On day 6 replace 50% of the culture media with new complete RPMI with 10 ng/ml mrGMCSF.
SECTION: Bone Marrow-derived Dendritic cells (BMDC, 0-9 days of experiment):
9. On day 9 collect all non-adherent and loosely adherent cells and count. Bring to 500,000 cell/ml in complete RPMI.
SECTION: Bone Marrow-derived Dendritic cells (BMDC, 0-9 days of experiment):
10. 3 mL of BMDC cells/ per well of a 6-well plate.  Add sonicate of H. pylori 100 μg/ml (300 µg/well) and incubate for 360 min at 37 °C. Leave at least two wells without bacterial sonicate for a control.

SECTION: Bone Marrow-derived Dendritic cells (BMDC, 0-9 days of experiment):
11. After 6 hours collect BMDC gently using cell scrapper and wash.
SECTION: Bone Marrow-derived Dendritic cells (BMDC, 0-9 days of experiment):
12. Pellet cells by spinning down at 800 x g, 5 min (Eppendorf 5810 – 2000 RPM).

SECTION: Bone Marrow-derived Dendritic cells (BMDC, 0-9 days of experiment):
13. Remove supernatant completely and add 2 mL of Room temperature (RT) 1% PFA and mix gently with a pipette. Leave at RT for 9 minutes.

SECTION: Bone Marrow-derived Dendritic cells (BMDC, 0-9 days of experiment):
14. Quench with 13 ml of 0.1M Glycine in complete RPMI (1 1M Glycine : 9 Complete RPMI).
SECTION: Bone Marrow-derived Dendritic cells (BMDC, 0-9 days of experiment):
15. Pellet cells by spinning down at 800 x g, 5 min and dumping the supernatant.
SECTION: Bone Marrow-derived Dendritic cells (BMDC, 0-9 days of experiment):
16. Repeat  -    two more times.
SECTION: Bone Marrow-derived Dendritic cells (BMDC, 0-9 days of experiment):
17. Resuspend in complete RMPI count and bring to 1x106 cells/ml .
SECTION: 2C CD8 T cells (day 9)
18. Collect spleens from 2C TCR transgenic mice of 6-8 weeks old, verify no presence of thymoma in the mouse.
SECTION: 2C CD8 T cells (day 9)
19. Mash spleen through a PBS pre-primed 70 µm cell strainer using a 3 ml syringe plunger. Wash strainer with additional 10 mL of PBS.

SECTION: 2C CD8 T cells (day 9)
20. Add 3 mL of ACK RBC lysis buffer to the splenocyte pellet and leave for 1 min, mix gently.

SECTION: 2C CD8 T cells (day 9)
21. Block lysis buffer with 12 mL of complete RPMI, count cells and take 100 µL aliquot for purification control (a) into a separate Eppendorf tube.

SECTION: 2C CD8 T cells (day 9)
22. Proceed with EasySep™ Mouse CD8+ T Cell Isolation Kit (Stemcell; Cat#19853) according to the manufacturer protocol. After spinning resuspend cell pellet in Stemcell Isolation Buffer 108/ml.
SECTION: 2C CD8 T cells (day 9)
23. Transfer cell to a 5 ml round bottom conical tube and add 20 µL of Fc Blocking reagent per 1 ml of cell suspension.
SECTION: 2C CD8 T cells (day 9)
24. Add 50 µl/ml of Isolation cocktail to cell suspension and incubate 20 min on ice.

SECTION: 2C CD8 T cells (day 9)
25. Add 125 µl/ ml of pre-vortexed for 30 s Rapid spheres and incubate 5 min at Room temperature

SECTION: 2C CD8 T cells (day 9)
26.  Top to 2.5 mL with Stemcell isolation buffer, transfer to EasySep™ Magnet and let stand for 3 min in the hood.

SECTION: 2C CD8 T cells (day 9)
27. Dump negatively selected CD8 T cells from the falcon tube in the magnet to a new 15 ml conical tube. Take a 50 µL aliquot of cell suspension for purification control (b).

SECTION: 2C CD8 T cells (day 9)
28. Top the 15 ml tube with PBS and spin down at 450 x g, 5 min(Eppendorf 5810 – 1500 rpm).

SECTION: 2C CD8 T cells (day 9)
29. Stain pelleted 2C CD8 T cells with 2 mL 2.5 µM cell tracker Tag-it Violet in PBS 12 min at 37 °C in the dark.

SECTION: 2C CD8 T cells (day 9)
30. Add 2 mL of the FBS and incubate an additional 5 min in the dark at Room temperature to efflux and quench excessive Tag-it violet dye.

SECTION: 2C CD8 T cells (day 9)
31. Wash cells three times with 10 ml of complete RPMI. 
SECTION: 2C CD8 T cells (day 9)
32. Resuspend in complete RMPI count and bring to 1x106 cells/ml.
SECTION: 2C CD8 T cells (day 9)
33. Take an aliquot of 500 µL Tag-it Violet-2C CD8 T cells and store in the fridge (a undiluted Tag-it violet signal, one of undivided cells control for flow cytometry).
SECTION: 2C CD8 T cells (day 9)
34. Stain purification controls (a) and (b) with antiCD8 and viability to access CD8 T cell purification quality.
SECTION: OT-1  CD8 T cells (TCR Tg CD8 T cell control)
35. collect inguinal, popliteal, axillar, and neck subcutaneous lymph nodes from OT-1 x Rag1 -/- mice. 

 

SECTION: OT-1  CD8 T cells (TCR Tg CD8 T cell control)
36. Mesh lymph nodes through a pre-primed with PBS 70 µm 1 ml syringe plunger.
SECTION: OT-1  CD8 T cells (TCR Tg CD8 T cell control)
37. Wash the strainer with 10 mL of PBS. Take 500 µL aliquot for CD8 frequency assessment using flow cytometry.

SECTION: OT-1  CD8 T cells (TCR Tg CD8 T cell control)
38. Spin cells down 450 x g, 5 min.

SECTION: OT-1  CD8 T cells (TCR Tg CD8 T cell control)
39. Stain pelleted OT-1 CD8 T cells with 2 ml 2.5 µM cell tracker Tag-it Violet in PBS 12 min at 37 °C in the dark.

SECTION: OT-1  CD8 T cells (TCR Tg CD8 T cell control)
40. Add 2 mL of the FBS and incubate an additional 5 min in the dark at Room temperature to efflux and quench excessive Tag-it violet dye.

SECTION: OT-1  CD8 T cells (TCR Tg CD8 T cell control)
41. Wash cells three times with complete RPMI.
SECTION: OT-1  CD8 T cells (TCR Tg CD8 T cell control)
42. Resuspend in complete RMPI count and bring to 1x106 cells/ml.
SECTION: CD4CD25- (CD4 conv) and CD4CD25 T (Tregs) (day 9)
43. Collect spleens of three Pink1+/+ and three Pink1-/- mice.
SECTION: CD4CD25- (CD4 conv) and CD4CD25 T (Tregs) (day 9)
44. Mash spleen individually through the 70 µm cell strainers and lyse RBC (as in step 20-21).
SECTION: CD4CD25- (CD4 conv) and CD4CD25 T (Tregs) (day 9)
45. Pull three spleens of each genotype together and count cells with hemocytometer and take a 100 µL aliquot for purification control (c). (Expected spleen count for B6.129 mixed genetic background mouse spleen is 5x107).

SECTION: CD4CD25- (CD4 conv) and CD4CD25 T (Tregs) (day 9)
46. Spin cells down at 450 x g, 5 min, resuspend pellets in 600 µL of MACS buffer per 1.5x108 (adjust the volume for more cells) and transfer to a new labeled 15 ml conical tube.

SECTION: CD4CD25- (CD4 conv) and CD4CD25 T (Tregs) (day 9)
47. Add 150 µL of the CD4+CD25 regulatory T cell Biotin-Antibody Cocktail (100 µl/ 400µl cell suspension) and incubate on ice 15 min.

SECTION: CD4CD25- (CD4 conv) and CD4CD25 T (Tregs) (day 9)
48. Wash cells by adding 10 mL of MACS buffer and spinning it down 450 x g, 5 min.
Resuspend pellets in 1200 mL of MACS buffer (800/ 108) and add 300 µL of anti-Biotin MicroBeads (200/108 cells) incubate 15 min on ice.

SECTION: CD4CD25- (CD4 conv) and CD4CD25 T (Tregs) (day 9)
49. Immediately transfer the QuadroMACS separator with two LD columns into the hood and prime columns with 2 mL of MACS buffer.

SECTION: CD4CD25- (CD4 conv) and CD4CD25 T (Tregs) (day 9)
50. Transfer cells to the columns and collect negatively selected unlabeled total CD4 T cells to a new 15 ml labeled conical tubes.
SECTION: CD4CD25- (CD4 conv) and CD4CD25 T (Tregs) (day 9)
51. Wash columns three times with 1 mL MACS buffer (passing through LD columns take a long time, make sure collection tubes under the columns is placed on ice).

SECTION: CD4CD25- (CD4 conv) and CD4CD25 T (Tregs) (day 9)
52. Take a 50 µL aliquot of total CD4 T cells in a separate Eppendorf cup – control (d). Spin cells down and add to the pellet 600 µL of MACS buffer with 15µl of anti CD25-PE.

SECTION: CD4CD25- (CD4 conv) and CD4CD25 T (Tregs) (day 9)
53. Incubate on ice 20 min in the dark.

SECTION: CD4CD25- (CD4 conv) and CD4CD25 T (Tregs) (day 9)
54. Top with 10 of MACS buffer, mix and take 100 µL aliquote (control e).

SECTION: CD4CD25- (CD4 conv) and CD4CD25 T (Tregs) (day 9)
55. After spinning cells down 450 x g, 5 min aspirate supernatant and resuspend cells in 1350 of buffer (900/108).

SECTION: CD4CD25- (CD4 conv) and CD4CD25 T (Tregs) (day 9)
56. Add 150 µL of anti-PE microbeads (100/108) and place on ice for another 15 min.

SECTION: CD4CD25- (CD4 conv) and CD4CD25 T (Tregs) (day 9)
57. Place 2 LS columns on the magnet and prime them with 3 ml buffer and discard the pass through.
SECTION: CD4CD25- (CD4 conv) and CD4CD25 T (Tregs) (day 9)
58. Transfer samples to the columns and collect the pass through into a new 15 ml conical tube on ice labeled CD4+CD25-.
SECTION: CD4CD25- (CD4 conv) and CD4CD25 T (Tregs) (day 9)
59. Wash column with 3 ml buffer three times and collect flow through. Take a 500 µL aliquot from the flow through for a control (f).

SECTION: CD4CD25- (CD4 conv) and CD4CD25 T (Tregs) (day 9)
60. Spin CD4 CD25- cells down 450 x g, 5 min and resuspend in 2 ml of complete RPMI with 1 µg/ml anti-CD3 (145-2c11) and place in the CO2 incubator to culture for 60 min at 37 °C.

SECTION: CD4CD25- (CD4 conv) and CD4CD25 T (Tregs) (day 9)
61. Simultaneously with the   transfer LS columns from the magnet to the new conical tube labeled CD4+CD25+.

SECTION: CD4CD25- (CD4 conv) and CD4CD25 T (Tregs) (day 9)
62. Add 3 mL of buffer to the LS column and use column plunger to isolate CD25+ cells.

SECTION: CD4CD25- (CD4 conv) and CD4CD25 T (Tregs) (day 9)
63. Replunge dry columns 5 times to completely recover the cells.
SECTION: CD4CD25- (CD4 conv) and CD4CD25 T (Tregs) (day 9)
64. Add 10 mL MACS buffer and spin down the cell.

SECTION: CD4CD25- (CD4 conv) and CD4CD25 T (Tregs) (day 9)
65. Completely remove the supernatant containing unbound microbeads and resuspend in 1 mL of complete media (take 30 µL aliquot for a control (g).

SECTION: CD4CD25- (CD4 conv) and CD4CD25 T (Tregs) (day 9)
66. Immediately acquire control f and g (10 µl only) on a cytometer, using PE channel if purity of control (g)- PE+ cells are lower than 85% repeat steps [ - and  - ] one more time. If control f has CD25+ cells more than 1% repeat steps [ - ] one more time.

SECTION: CD4CD25- (CD4 conv) and CD4CD25 T (Tregs) (day 9)
68. Spin plate down and stain viability with FVS780 in PBS dilution 1:2000 50 µl per well, 15 min on ice in the dark.

SECTION: CD4CD25- (CD4 conv) and CD4CD25 T (Tregs) (day 9)
67. Transfer controls d-g to a plate for staining with FoxP3 antibody.
SECTION: CD4CD25- (CD4 conv) and CD4CD25 T (Tregs) (day 9)
69. Wash viability stain with 300 µL of MACS buffer and leave cells for fixation in FoxP3 fixing buffer in the fridge 120 µl/well.

SECTION: CD4CD25- (CD4 conv) and CD4CD25 T (Tregs) (day 9)
70. After an hour take CD4+CD25- cells from the incubator and top the tube with PBS.
SECTION: CD4CD25- (CD4 conv) and CD4CD25 T (Tregs) (day 9)
71. Spin CD4+CD25- cells down and resuspend in 2 ml of CFSE 1.25 µM stain in the same manner as B12-B14.
SECTION: CD4CD25- (CD4 conv) and CD4CD25 T (Tregs) (day 9)
72. While staining CD4+CD25- count CD4CD25+ and bring them to the concentration of 106 cells/ml in complete RPMI.
SECTION: CD4CD25- (CD4 conv) and CD4CD25 T (Tregs) (day 9)
73. Resuspend CFSE-CD4+CD25 in 3 ml complete RPMI, take 100 µL aliquote for undilute CFSE staining control and place it in the fridge.

SECTION: CD4CD25- (CD4 conv) and CD4CD25 T (Tregs) (day 9)
74. using CD4+CD25+ Regulatory T Cell Isolation Kit (Miltenyi Biotech; Cat #130-091-041) according to manufacturer’s protocol. CD4 conv T cells were resuspended in complete RPMI 10% FBS and incubated for 60 min at 37 °C with 1 µg/ml of anti-mouse CD3 (145-2c11 clone; Biolegend; Cat#100363) or left untreated. Cells were washed in PBS and stained with CFSE 1.25 µM in the same manner as Tag-it violet staining.

SECTION: CD4CD25- (CD4 conv) and CD4CD25 T (Tregs) (day 9)
75. Purity of magnetic selection of 2C CD8, CD4+CD25+ and CD4+CD25- cells was determined with flow cytometry prior to co-culture. Regulatory CD25+CD4 T cells were stained with anti-FoxP3 and acquired with flow cytometry.
SECTION: CD4CD25- (CD4 conv) and CD4CD25 T (Tregs) (day 9)
76. Co-culture was performed in 10% FBS complete RPMI in 96-well round bottom plates for suspension cells (Sarstedt; Cat#83.3925.500) at a following ratio: 50,000 2C to 50,000 CD4 conv cells to 100,000 fixed BMDC. For suppression assay Tregs were added to the co-culture at (1-0.25):1 2C CD8 T ratio. Each condition was performed in technical replicates. At 24- and 72-hours cells were collected and assessed by flow cytometry for Activation Induced Markers expression and proliferation.
SECTION: CD4CD25- (CD4 conv) and CD4CD25 T (Tregs) (day 9)
77. Co-culture conditions for proliferation assay (excluding single cell type controls, and cell tracker controls from the fridge): 
  
 
 
SECTION: CD4CD25- (CD4 conv) and CD4CD25 T (Tregs) (day 9)
78. Suppression of proliferation was calculated using the following formula:

% suppression = 100x(1-% of divided 2C in the presence of Treg/ -% of divided 2C  at No Treg condition)