Immunophenotyping of human tumor-reactive T cell functionality using flow cytometry
MBTP 61 = Miltenyi Biotec-tested panel 61
MBTP 61 = Miltenyi Biotec-tested panel 61
This application protocol describes the analysis of human tumor-reactive T cell functionality by detection of intracellular and extracellular markers. To demonstrate the state-of-the-art panel, tumor-infiltrating leukocytes (TILs) were isolated from a human melanoma digest. The contained tumor-reactive T cells (TRTs) were enriched using CD137 MicroBeads and expanded for two weeks using the CliniMACS Prodigy®. For functionality testing, TRTs were then co-cultured with autologous or mismatched tumor cell lines in presence of a protein transport inhibitor containing monensin and brefeldin A. TRTs were subsequently analyzed by flow cytometry to determine their anti-tumor reactivity in terms of T cell degranulation (CD107a/LAMP-1), T cell activation (CD154, CD137), and production of inflammatory cytokines (IFNγ, TNFα).
Protocol
Surface staining
- 2×105 cells/well from autologous or mismatched tumor cell lines were seeded in a 96-well flat bottom plate in 100 µL TexMACS GMP Medium supplemented with 3% AB serum + 1% P/S.
- 2×105 tumor-reactive T cells/well were added to the autologous and mismatched tumor cell lines in 30 µL TexMACS GMP Medium supplemented with 3% AB serum + 1% P/S for a final volume of 130 µL/well.
- CD107a-VioBlue was added to each well according to the dilution mentioned in the respective data sheet and incubated at 37 °C and 5% CO2.
▲Note: As expression of CD107a is extremely transient due to recycling of granules, CD107a has to be stained during the stimulation culture. - BD GolgiPlug™ and BD GolgiStop™ were added to the culture in 20 µL TexMACS GMP Medium supplemented with 3% AB serum + 1% P/S after 1−2 hours for a final volume of 150 µL/well.
- Incubation was continued for a total maximal incubation time of 16−18 hours.
- Cells were harvested and centrifuged at 300×g for 5 minutes. Supernatant was aspirated completely.
- A staining cocktail with CD4-FITC and CD8-VioGreen was prepared according to the dilutions mentioned in the respective data sheets.
- 50 µL staining cocktail was added to the cells.
- Cells were resuspended and incubated for 10 minutes in the dark at room temperature (19−25 °C).
- Cells were washed by addition of 150 µL of PEB buffer and centrifuged at 300×g for 5 minutes. Supernatant was aspirated completely.
- Cells were resuspended in 50 µL of PEB buffer and 50 µL of Inside Fix.
- Cells were incubated for 20 minutes in the dark at room temperature (19−25 °C).
- Cells were washed by addition of 200 µL of PEB buffer and centrifuged at 300×g for 5 minutes. Supernatant was aspirated completely.
- Cells were washed with 200 µL of Inside Perm and centrifuged at 300×g for 5 minutes. Supernatant was aspirated completely.
- A staining cocktail with CD154-APC, TNFα Antibody-PE-Vio 770, IFNγ Antibody-APC-Vio 770, and CD137-PE was prepared according to the dilutions mentioned in the respective data sheets. Inside Perm was used to dilute the antibodies instead of PEB buffer.
- 50 µL staining cocktail was added to the cells.
- Cells were resuspended and incubated for 10 minutes in the dark at room temperature (19−25 °C).
- Cells were washed by addition of 150 µL of Inside Perm and centrifuged at 300×g for 5 minutes. Supernatant was aspirated completely.
- Cells were resuspended in 150 µL of PEB buffer before flow cytometric analysis using the MACSQuant 10 Analyzer.
Antibody panel for human tumor-reactive T cell functionality
| Specificity | Clone | Purpose | Fluorochrome | Detection filter (nm) (laser) |
| CD4 | REA623 | Helper T cells | FITC | 525/50 (blue) |
| CD8 | REA734 | Cytotoxic T cells | VioGreen™ | 525/50 (violet) |
| IFNγ | REA600 | Inflammatory cytokine | APC-Vio® 770 | 750 LP (red) |
| CD154 | REA238 | T cell activation | APC | 655–730 (red) |
| CD107a/LAMP-1 | REA792 | T cell degranulation | VioBlue® | 450/50 (violet) |
| CD137 | REA765 | T cell activation | PE | 585/40 (blue) |
| TNFα | cA2 | Inflammatory cytokine | PE-Vio 770 | 750 LP (blue) |
Gating strategy
View details
Materials
Antibody panel for human tumor-reactive T cell functionality
| Specificity | Clone | Purpose | Fluorochrome | Detection filter (nm) (laser) |
| CD4 | REA623 | Helper T cells | FITC | 525/50 (blue) |
| CD8 | REA734 | Cytotoxic T cells | VioGreen™ | 525/50 (violet) |
| IFNγ | REA600 | Inflammatory cytokine | APC-Vio® 770 | 750 LP (red) |
| CD154 | REA238 | T cell activation | APC | 655–730 (red) |
| CD107a/LAMP-1 | REA792 | T cell degranulation | VioBlue® | 450/50 (violet) |
| CD137 | REA765 | T cell activation | PE | 585/40 (blue) |
| TNFα | cA2 | Inflammatory cytokine | PE-Vio 770 | 750 LP (blue) |
Additional reagents used
- PEB buffer: PBS, pH 7.2, 0.5% bovine serum albumin (BSA), and 2 mM EDTA
- Inside Stain Kit (# 130-090-477)
- TexMACS GMP Medium (# 170-076-306)
- AB serum
- Penicillin/streptomycin (P/S)
- BD GolgiPlug™
- BD GolgiStop™
- Flat bottom 96-well plate
- Flow cytometer, for example, MACSQuant® Analyzer 10 (# 130-096-343)
- (Optional) MACSQuant Calibration Beads (# 130-093-607)
- (Optional) MACS® Comp Bead Kit, anti-REA (# 130-104-693)
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