Sep 22, 2024
Mitochondrial antigen presentation (MitAP) to primary 2C CD8 T (proliferation and suppression) assay
- Alexandra Kazanova1,
- Samantha Gruenheid1
- 1McGill Research Centre on Complex Traits and Department of Microbiology and Immunology, McGill University
Protocol Citation: Alexandra Kazanova, Samantha Gruenheid 2024. Mitochondrial antigen presentation (MitAP) to primary 2C CD8 T (proliferation and suppression) assay. protocols.io https://dx.doi.org/10.17504/protocols.io.dm6gpzk25lzp/v1
Manuscript citation:
Modeling gene-environment interactions in Parkinson’s Disease: Helicobacter pylori infection of Pink1−/− mice induces CD8 T cell-dependent motor and cognitive dysfunction
Alexandra Kazanova, Jacqueline Sung, Nathalia Oliveira, Christina Gavino, Sherilyn Recinto, Hicham Bessaiah, Jessica Pei, Lindsay Burns, Willemein Miller, Morgane Brouillard-Galipeau, Lei Zhu, Lucia Guerra, Moustafa Nouh Elemeery, Adam MacDonald, Joel Lanoix, Pierre Thibault, Heidi McBride, Michel Desjardins, Jo Anne Stratton, Nathalie Labrecque, Samantha Gruenheid
bioRxiv 2024.02.25.580545; doi: https://doi.org/10.1101/2024.02.25.580545
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: September 17, 2024
Last Modified: September 22, 2024
Protocol Integer ID: 108061
Keywords: ASAPCRN
Funders Acknowledgement:
Aligning Science Across Parkinson’s (ASAP)
Grant ID: ASAP 000525
Abstract
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.
Materials
Complete RPMI 1640:
| A | B | |
| RPMI 1640 with Glutamax (Gibco) | with 10% FBS | |
| sodium pyruvate | 1% | |
| Hepes | 1 mM | |
| penicillin/streptomycin | 1% |
MACS buffer: PBS, 2% FBS and 2mM EDTA
StemCell Isolation Buffer: PBS, 2% FBS and 1 mM EDTA
ACK (Ammonium-Chloride-Potassium) red blood cell lysis buffer: 8.29g NH4Cl, 1g KHCO3, 0.0367g EDTA, distilled water to 1L)
090-150- FBS, Premium Canadian Origin, 500mLWisent BioproductsCatalog #090-150
PBS 10x (Sigma; Cat# P5493-4L)Phosphate buffered salineMerck MilliporeSigma (Sigma-Aldrich)Catalog #P5493-4L without calcium/magnesium
Recombinant Mouse GM-CSF (carrier-free)BioLegendCatalog #576308
Purified anti-mouse CD3ε AntibodyBioLegendCatalog #100302
EasySep™ Mouse CD8+ T Cell Isolation Kit For processing 1 x 10^9 cells
STEMCELL Technologies Inc.Catalog #19853
EasySep™ Magnet For isolating 2.5 x 10^8 cells
STEMCELL Technologies Inc.Catalog #18000
CD4+CD25+ Regulatory T Cell Isolation Kit, mouseMiltenyi BiotecCatalog #130-091-041
CFSE Cell Division Tracker KitBioLegendCatalog #423801
eBioscience™ Fixable Viability Dye eFluor™ 780Thermo FisherCatalog #65-0865-18
Anti-mouse CD8a; -FoxP3; CD4; CD25 conjugated to selected fluorochromes (except PE).
e-Bioscience Foxp3 / Transcription Factor Staining Buffer SetInvitrogen - Thermo FisherCatalog # 00-5523-00
MACS® LD and LS Columns Catalog #130-042-401, Catalog #130-042-901
QuadroMACS™ Separators
Tag-it Violet Biolegend Cat#455101
H. Pylori PMSS1 strain sonicate
70 µm cell strainers
Paraformaldehyde 1%
1M glycine in PBS
Mice
Pink1+/+ (n=6) and Pink1-/- mice (n=6). 2C TCR transgenic mouse (n=1-2). OT-1xRAG1-/- (n=1-2).
Bone Marrow-derived Dendritic cells (BMDC, 0-9 days of experiment):
Bone Marrow-derived Dendritic cells (BMDC, 0-9 days of experiment):
1m
1m
Femurs from three Pink1+/+ and three Pink1-/- mice are cleaned manually from soft tissues and flushed with ice cold PBS.
Cells are pelleted and 2 mL of ACK RBC lysis buffer are added for 00:01:00 and mixed gently with 1 ml pipette tip.
1m
Lysis buffer is blocked with 13 mL of complete RPMI and pelleted with centrifugation.
Cells are resuspended, counted with hemocytometer and brough to 200,000 cell/ml in complete RPMI.
10 ng/ml of mr-GMCSF is added to BMDC for differentiation.
Cells are plated in non-tissue culture treated plasticware (6 well plate, sarstedt) for 72:00:00 at 37 °C .
3d
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).
On day 6 replace 50% of the culture media with new complete RPMI with 10 ng/ml mrGMCSF.
On day 9 collect all non-adherent and loosely adherent cells and count. Bring to 500,000 cell/ml in complete RPMI.
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 06:00:00 at 37 °C . Leave at least two wells without bacterial sonicate for a control.
6h
After 6 hours collect BMDC gently using cell scrapper and wash.
Pellet cells by spinning down at 800 x g, 00:05:00 (Eppendorf 5810 – 2000 RPM).
5m
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.
Quench with 13 ml of 0.1M Glycine in complete RPMI (1 1M Glycine : 9 Complete RPMI).
Pellet cells by spinning down at 800 x g, 00:05:00 and dumping the supernatant.
5m
Repeat go to step #13 - go to step #15 two more times.
Resuspend in complete RMPI count and bring to 1x106 cells/ml .
2C CD8 T cells (day 9)
2C CD8 T cells (day 9)
Collect spleens from 2C TCR transgenic mice of 6-8 weeks old, verify no presence of thymoma in the mouse.
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.
Add 3 mL of ACK RBC lysis buffer to the splenocyte pellet and leave for 00:01:00 , mix gently.
1m
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.
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.
Transfer cell to a 5 ml round bottom conical tube and add 20 µL of Fc Blocking reagent per 1 ml of cell suspension.
Add 50 µl/ml of Isolation cocktail to cell suspension and incubate 00:20:00 On ice .
20m
Add 125 µl/ ml of pre-vortexed for 00:00:30 Rapid spheres and incubate 00:05:00 at Room temperature
5m 30s
Top to 2.5 mL with Stemcell isolation buffer, transfer to EasySep™ Magnet and let stand for 00:03:00 in the hood.
3m
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).
Top the 15 ml tube with PBS and spin down at 450 x g, 00:05:00 (Eppendorf 5810 – 1500 rpm).
5m
Stain pelleted 2C CD8 T cells with 2 mL 2.5 µM cell tracker Tag-it Violet in PBS 00:12:00 at 37 °C in the dark.
12m
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.
Wash cells three times with 10 ml of complete RPMI.
Resuspend in complete RMPI count and bring to 1x106 cells/ml.
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).
Stain purification controls (a) and (b) with antiCD8 and viability to access CD8 T cell purification quality.
OT-1 CD8 T cells (TCR Tg CD8 T cell control)
OT-1 CD8 T cells (TCR Tg CD8 T cell control)
collect inguinal, popliteal, axillar, and neck subcutaneous lymph nodes from OT-1 x Rag1 -/- mice.
Note
Make sure not to take any fat tissue.
Mesh lymph nodes through a pre-primed with PBS 70 µm 1 ml syringe plunger.
Wash the strainer with 10 mL of PBS. Take 500 µL aliquot for CD8 frequency assessment using flow cytometry.
Spin cells down 450 x g, 00:05:00 .
5m
Stain pelleted OT-1 CD8 T cells with 2 ml 2.5 µM cell tracker Tag-it Violet in PBS 00:12:00 at 37 °C in the dark.
12m
Add 2 mL of the FBS and incubate an additional 00:05:00 in the dark at Room temperature to efflux and quench excessive Tag-it violet dye.
5m
Wash cells three times with complete RPMI.
Resuspend in complete RMPI count and bring to 1x106 cells/ml.
CD4CD25- (CD4 conv) and CD4CD25 T (Tregs) (day 9)
CD4CD25- (CD4 conv) and CD4CD25 T (Tregs) (day 9)
3h 40m
3h 40m
Collect spleens of three Pink1+/+ and three Pink1-/- mice.
Mash spleen individually through the 70 µm cell strainers and lyse RBC (as in step 20-21).
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).
Spin cells down at 450 x g, 00:05:00 , 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.
5m
Add 150 µL of the CD4+CD25 regulatory T cell Biotin-Antibody Cocktail (100 µl/ 400µl cell suspension) and incubate On ice 00:15:00 .
15m
Wash cells by adding 10 mL of MACS buffer and spinning it down 450 x g, 00:05:00 .
Resuspend pellets in 1200 mL of MACS buffer (800/ 108) and add 300 µL of anti-Biotin MicroBeads (200/108 cells) incubate 00:15:00 On ice .
20m
Immediately transfer the QuadroMACS separator with two LD columns into the hood and prime columns with 2 mL of MACS buffer.
Transfer cells to the columns and collect negatively selected unlabeled total CD4 T cells to a new 15 ml labeled conical tubes.
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 ).
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.
Incubate On ice 00:20:00 in the dark.
20m
Top with 10 of MACS buffer, mix and take 100 µL aliquote (control e).
After spinning cells down 450 x g, 00:05:00 aspirate supernatant and resuspend cells in 1350 of buffer (900/108).
5m
Add 150 µL of anti-PE microbeads (100/108) and place On ice for another 00:15:00 .
15m
Place 2 LS columns on the magnet and prime them with 3 ml buffer and discard the pass through.
Transfer samples to the columns and collect the pass through into a new 15 ml conical tube on ice labeled CD4+CD25-.
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).
Spin CD4 CD25- cells down 450 x g, 00:05:00 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 01:00:00 at 37 °C .
1h 5m
Simultaneously with the go to step #60 transfer LS columns from the magnet to the new conical tube labeled CD4+CD25+.
Add 3 mL of buffer to the LS column and use column plunger to isolate CD25+ cells.
Replunge dry columns 5 times to completely recover the cells.
Add 10 mL MACS buffer and spin down the cell.
Completely remove the supernatant containing unbound microbeads and resuspend in 1 mL of complete media (take 30 µL aliquot for a control (g).
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 [go to step #57 -go to step #59 and go to step #61 -go to step #63 ] one more time. If control f has CD25+ cells more than 1% repeat steps [ -go to step #60 ] one more time.
Transfer controls d-g to a plate for staining with FoxP3 antibody.
Spin plate down and stain viability with FVS780 in PBS dilution 1:2000 50 µl per well, 00:15:00 On ice in the dark.
15m
Wash viability stain with 300 µL of MACS buffer and leave cells for fixation in FoxP3 fixing buffer in the fridge 120 µl/well.
After an hour take CD4+CD25- cells from the incubator and top the tube with PBS.
Spin CD4+CD25- cells down and resuspend in 2 ml of CFSE 1.25 µM stain in the same manner as B12-B14.
While staining CD4+CD25- count CD4CD25+ and bring them to the concentration of 106 cells/ml in complete RPMI.
Resuspend CFSE-CD4+CD25 in 3 ml complete RPMI, take 100 µL aliquote for undilute CFSE staining control and place it in the fridge.
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 01:00:00 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.
1h
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.
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.
Co-culture conditions for proliferation assay (excluding single cell type controls, and cell tracker controls from the fridge):
| A | B | C | D | E | F | G | H | I | |
| BMDC | H. pylori for BMDC | CD4 conv | aCD3 for CD4 | OT-1 CD8T | 2C CD8 T | SIINFEKL | SYIRYYGL | Function | |
| + | - | - | - | + | - | + | - | OT-1 proliferation – positive control | |
| + | - | - | - | + | - | - | - | OT-1 proliferation – negative control | |
| + | - | - | - | - | + | - | + | 2C proliferation – positive control | |
| + | - | - | - | - | + | - | - | 2C proliferation – negative control | |
| + | + | + | + | - | + | - | - | 2C proliferation - experimental condition | |
| + | + | + | + | + | - | - | - | TCR 2C specificity control | |
| + | - | + | + | - | + | - | - | 2C No proliferation – infection effect on MitAP control | |
| + | + | + | - | - | + | - | - | CD4 help effect control | |
| + | + | - | - | - | + | - | - | CD4 help effect control | |
| - | - | + | + | + | - | - | - | Bystander activation control OT1 | |
| - | - | + | + | - | + | - | - | Bystander activation control 2C | |
| + | + | + | + | - | - | - | - | CD4 proliferation | |
| + | - | + | + | - | - | - | - | CD4 proliferation | |
| - | - | + | + | - | - | - | - | CD4 proliferation | |
| - | - | + | - | - | - | - | - | CD4 proliferation negative control |
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)