T-cells recognize peptides/MHC complexes (pMHC) through the T-cell receptor (TCR). This is the first step for the initiation and shaping of protective immunity against viruses and tumor antigens. Fluorescently labelled pMHC tetramers have dramatically transformed the detection of antigen specific T-cells. MHC tetramer staining of antigen specific T cell clonotypes is detected by flow cytometry to probe T-cell responses1 and to further characterize antigen-specific T cells, for example, to study the surface markers as well as intracellular proteins. The measurement and analysis of effector function of antigen-reactivated T cells is now possible by MHC tetramer and flow cytometry-based intracellular cytokine staining (ICS). This method allows concurrent phenotypic characterization and cytokine detection within single cells2.
The production of cytokines plays an important role in immune response. Cytokines are involved in many pathways, including the induction of many anti-viral proteins by IFNγ, the induction of T cell proliferation by IL-2, and the inhibition of viral gene expression and replication by TNFα3. Cytokines are not preformed factors but are produced and secreted in response to cellular activation.
Intracellular cytokine staining is a very useful and widely used flow cytometry-based assay, which detects the production and accumulation of cytokines after cell stimulation. Combining intracellular cytokine staining with other flow cytometry protocols for immunotyping using cell surface markers or with MHC tetramers to detect antigen-specific responses4 can make it a flexible and versatile method.
Note: This is a general protocol for intracellular staining with MHC tetramers. The list of reagents provided are only examples. The protocol below is for staining in a 96-well plate format, 500,000 human PBMCs per well. Other formats will likely require adjusting reagent volumes.
- Add Brefeldin A (final concentration of 5 µg/mL) and monensin (final concentration of 0.7 µg/mL) to stimulated PBMC and incubate for 4 hours.
- 15 minutes prior to spinning down cells (3 hours 45 minutes after Brefeldin A/monenin), add 50 mM Dasatinib in cell culture media.
- Spin cells at 500 x g and decant.
- Wash twice with 200 µL PBS to get rid of serum. Spin cells at 500 x g and decant between each wash.
- Add 50 µL of cell surface master mix (composition listed below) and incubate for 30min at room temperature in the dark.
- CD3 (0.25 µL per sample)
- CD8 (0.5 µL per sample)
- MBL tetramer (2 µL per sample)
- Live/Dead stain (1:200 dilution for 500,000 cells)
- Dasatinib in PBS at a final concentration of 50 nM
- FACS incubation buffer (0.5% BSA)
- Wash with PBS two times (150 µL for the first wash and 200 µL for the second wash. Spin cells at 500 x g and decant between each wash.
- Add 200 µL fixation buffer and pipette to mix. Incubate for 45 minutes at room temperature in the dark.
- Spin cells at 700 x g and decant
- Re-suspend cells in permeabilizing buffer and pipette to mix.
- Incubate for 10 minutes at room temperature in the dark.
- Wash twice with 200 µL permeabilizing buffer. Spin cells at 700 x g and decant between each wash.
- Add 50 µL of intracellular master mix and incubate for 30min at room temperature in the dark.
- IFNγ (1:100 dilution for 500,000 cells)
- Permeabilizing buffer
- Wash with permeabilizing buffer two times (150 µL for the first wash and 200 µL for the second wash). Spin cells at 7500 x g and decant between each wash.
- Resuspend in 200 µL PBS for flow analysis.
|Product Code||Product Name|
|4844||IMMUNOCYTO CD107a Detection Kit|
|FP10255010||Anti-CD3 (Human) mAb-FITC (Monoclonal Antibody)|
|FP10588010||Anti-CD8a (Human) mAb-APC (Monoclonal Antibody)|
- Su, L., del Alcazar, D., Stelekati, E., Wherry, E., Davis, M., (2016) Antigen Exposure Shapes the Ratio between Antigen-Specific Tregs and Conventional T Cells in Human Peripheral blood, PNAS, 10 (1073), 6192-6198, doi: 10.1073/pnas.1611723113
- Pastore, G., Carraro, M., Pettini, E., Nolfi, E., Medaglini, D. Ciabattini, A., (2019) Optimized Protocol for the Detection of Multifunctional Epitope-Specific CD4+ T Cells Combining MHC-II tetramer and Intraceullar Cytokine Staining Technologies, Frontiers in Immunology, 10(2304), 1-10, doi: 10.3389/fimmu.2019.02304
- Appay, V., Nixon, D., Donahoe, S., Gillespie, G., Tong, T., King, A., Ogg, G., Spiegal, H., Conlon, C., Spina, C., Havlir, D., Richman, D., Waters, A., Eaterbrook, P., McMichael, A., Rowland-Jones, S., (2000) HIV-specific CD8+ T Cells Produce Antiviral Cytokines but are Impaired in Cytolytic Function, Journal of Experimental Medicine, 192 (1), 63-75, doi: 10.1084/jem/192.1.63
- Aloulou, M., Carr, E., Gador, M., Bignon, A., Liblau, R., Fazilleau, N., Linterman, M., (2016) Follicular Regulatory T Cells can be Specific for the Immunizing Antigen and Derive from Native T Cells, Nature Communications, 7 (10579), 1-10, doi: 10:1038/ncomms10579