Evaluation of antigen binding by CD8+ T cells after in vivo treatment with myeloid derived suppressor cells

doi:10.1038/nprot.2007.240

Method Article

Evaluation of antigen binding by CD8+ T cells after in vivo treatment with myeloid derived suppressor cells

https://doi.org/10.1038/nprot.2007.240

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Cell biology

Developmental biology

Biological techniques

T cells

pMHC binding

This protocol is adapted from previous work of Dr. Schneck and used to evaluate binding of OT-1 and 2C specific pMHC by CD8+ T cells.

ACK lysing buffer 8.29g NH₄Cl (0.15M), 1g KHCO₃(10.0mM), 37.2 mg Na₂EDTA(0.1mM) Add 800ml H₂0 and adjust pH to 7.2-7.4, Add H₂0 to 1L. Filter sterilize and store at RT.

Wash buffer PBS+2%FBS+.05% azide

Dimers SIINKb-Ig and SIYKb-Ig dimmers

Biotin-Gr-1 antibody (Pharmingen, CA)

Strepatavidin beads (Miltenyi biotech, CA)

Specific peptide SIINFEKL and incomplete Freund’s adjuvant

T cell column (R & D systems, MN)

Magnetic Columns (Miltenyi biotech, CA)

Centrifuge with temperature control

Flow cytometer (BD FacsCalibur)

Flow analysis software Flowjo vers 8.2

Day 1: Isolate T cells from OT-1 mice using T cell enrichment column and transfer 5-6 X 10⁶ cells intravenously into CD45.1 mice.

Day 3. Isolate MDSC from 3 week tumor-bearing mice (EL4 or MC38) using biotin-Gr-1 and streptavidin beads (Miltenyi biotech, CA) antibody and transfer 4-5 X 10⁶ cells intravenously into mice which have received OT-1 T cells. Immunize mice with SIINFEKL peptide 100 ug in 100 ul PBS plus 100 ul of incomplete Freund’s adjuvant.

Day 10:

Harvest spleens from mice and prepare a single cell suspension.
Pellet the cells by centrifugation (1700 RPM 5 min) at room temperature (RT) and aspirate the supernatant.
Resuspend the pellet in 5 ml/spleen of ACK Lysis Buffer.
Incubate at RT for 4-5 minutes with occasional shaking.
Stop the reaction by adding 40-50 μl of PBS.
Wash the cells with PBS twice (1700 RPM 5 min) at RT.
Use Cy5 (Molecular probes, OR) labeled SIINKb-Ig and SIYKb-Ig dimers as specific and non-specific MHC-Ig ligands, respectively.
Prepare a serial dilution (2000 nM – 12.5 nM) of 20 μl of Cy5 labeled MHC-Ig dimer in a FACS tube.
Adjust cells to a concentration of 10⁷ cells/ml in FACS wash buffer
Add 10 ul aliquot of cells to the FACS tube with the MHC-Ig dimer.
Incubate cells with the MHC-Ig dimers for 60-90 min on ice.
Without any washing add FITC labeled anti-CD8 and PE labeled anti-CD45.1 antibodies to each aliquot.
Incubate cells for an additional 20 min on ice and wash two times before flow cytometric analysis.
Specific MHC-Ig staining is calculated by subtracting the non-specific MHC-Ig staining from the total MHC-Ig staining within the population of CD45.1 negative CD8 positive cells.

Approximately 11 days

See accompanied paper

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Evaluation of antigen binding by CD8+ T cells after in vivo treatment with myeloid derived suppressor cells

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Evaluation of antigen binding by CD8+ T cells after in vivo treatment with myeloid derived suppressor cells

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Introduction

Reagents

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Timing

Anticipated Results

Associated Publications

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