Stimulation of Antigen-Specific T Cells Using Peptide Pools
Peptide pools are mixtures of short peptide sequences that, together, span the entire length of a protein (e.g. the SARS-CoV-2 Spike Protein) or represent key immunodominant epitopes (e.g. CEF (HLA Class I Control) —containing viral epitopes from cytomegalovirus, Epstein-Barr, and influenza) that can be used to stimulate antigen-specific CD4+ and/or CD8+ T cells. Activated T cells release downstream cytokines and upregulate activation markers that enable the antigen-specific cells to be detected and quantified, or isolated for analysis. By providing a simple, efficient, and cost-effective method for screening antigens and evaluating T cell responses, peptide pools are powerful tools that can be utilized for a broad range of applications, including vaccine development, immune cell monitoring/surveillance, and diagnostic assay development. Explore our complete product listing to find the right peptide pool for your research.
In this protocol, you will learn how to prepare peptide pool stocks and suspensions of peripheral blood mononuclear cells (PBMCs), and how to perform in vitro stimulation and detection of antigen-specific T cells using flow cytometry and ELISpot assays.
Materials
Preparing Peptide Pool Stocks
- Peptide pools of choice, e.g.:
- CEF (HLA Class I Control) Peptide Pool (Catalog #100-0675)
- SARS-CoV-2 (Spike Protein) Peptide Pool (Catalog #100-0676)
- DMSO
- Tissue culture grade water
- Tubes for preparing aliquots
Preparation of Cells
- Suspension of PBMCs
- Option A: Human Whole Peripheral Blood (Catalog #70504)
- Density gradient medium (e.g. Lymphoprep™, Catalog #07801) OR
- EasySep™ Direct Human PBMC Isolation Kit (Catalog #19654)
- Option B: Human Peripheral Blood Leukopak, Fresh (Catalog #70500)
- Ammonium Chloride Solution (Catalog #07800)
- Option A: Human Whole Peripheral Blood (Catalog #70504)
- ImmunoCult™-XF T Cell Expansion Medium (Catalog #10981) or cell culture medium of choice
Stimulation and Detection of Antigen-Specific T Cells
- ImmunoCult™-XF T Cell Expansion Medium (Catalog #10981) or cell culture medium of choice
- 24-Well Flat-Bottom Plate, Tissue Culture-Treated (Catalog #38017 OR #38021)
- Brefeldin A (Catalog #73012)
- Round-Bottom Polypropylene Tubes, 5 mL (Catalog #38056) or 96-Well Round-Bottom Microplates (Catalog #38018) for staining
- GloCell™ Fixable Viability Dye Red 780 (Catalog #75007)
Protocol
Part I: Preparation of Peptide Pool Stocks
The majority of our peptide pools are provided at ~25 µg per peptide (15 nmol) for the stimulation of up to 2.5 x 108 cells unless stated otherwise.
- Warm vial to room temperature (15 - 25°C). Centrifuge vial before opening.
- Add a small amount of pure DMSO (e.g. 40 µL). Vortex to mix.
Ensure the peptides are completely dissolved. Solubility can be facilitated by careful warming (< 40°C) or sonication.
- Dilute with sterile tissue-culture grade water to the desired concentration, e.g. for a 25 µg per peptide vial, add 210 µL of water (for a final volume of 250 µL, 210 µL water + 40 µL DMSO) to prepare a 100 µg/mL per peptide stock. Mix thoroughly.
- If not used immediately, prepare single-use aliquots and store at ≤ -20°C. Protect from direct light.
Part II: Preparation of Cells
Prepare a suspension of PBMCs from human whole peripheral blood (Method A) or a fresh human peripheral blood leukopak (Method B).
A. Human Whole Peripheral Blood
- Centrifuge human whole peripheral blood over a density gradient medium (e.g. Lymphoprep™, Catalog #07801). See full protocol available here.
- Isolate PBMCs using EasySep™ Direct Human PBMC Isolation Kit (Catalog #19654). See Product Information Sheet for protocol.
OR
B. Human Peripheral Blood Leukopak, Fresh
- Lyse red blood cells (RBCs) with Ammonium Chloride Solution (Catalog #07800) and remove platelets. See full protocol available here.
PBMCs should be resuspended in ImmunoCult™-XF T Cell Expansion Medium or the cell culture medium of choice at the required cell density for the protocol of choice. See below for some examples. PBMCs may be cryopreserved using CryoStor® CS10 (Catalog #100-1061) prior to use.
Enriched T cells with antigen-presenting cells (e.g. dendritic cells) can also be used for certain assays. Refer to our Technical Bulletin, Dendritic Cell/CD8+ T Cell Co-Culture to Assess Antigen-Specific T Cell Functionality, for more information.
Part III: Stimulation and Detection of Antigen-Specific T Cells
Reconstituted peptide pools can be further diluted in cell culture media (e.g. ImmunoCult™-XF T Cell Expansion Medium). A final concentration of ≥ 1 µg/mL per peptide is generally recommended for antigen-specific stimulation.
The final concentration of DMSO must be below 1% (v/v) to avoid toxicity in the biological system.
In your experiment, always include a negative control(s) (e.g. DMSO and/or Human (Actin) Peptide Pool) and positive control(s) (e.g. Phorbol 12-myristate 13-acetate (PMA; Catalog #74042), Ionomycin (Catalog #73722), and/or CEF (HLA Class I Control) Peptide Pool).
Intracellular Cytokine Staining for Flow Cytometry
- Starting with the peptide pool stock(s), prepare 10X (e.g. 10 µg/mL per peptide) working solution(s) in the cell culture medium of choice.
- Add 1 x 107 PBMCs in 900 µL of cell culture medium into wells of a 24-well tissue culture plate.
- Add 100 µL of 10X peptide pool working solution(s) to each well.
- Incubate the cells with peptide at 37°C, 5% CO2 for the desired incubation time. Typically 5 - 6 hours is recommended, but this should be optimized for your experiment.
After 2 hours, add Brefeldin A (Catalog #73012) to block cytokine secretion and return the plate to the incubator.
- After the incubation time is complete, harvest the cells by gently pipetting up and down to ensure that they are in suspension, and transfer the cells to an appropriate tube.
- Stain with a fixable viability dye (e.g. GloCell™ Fixable Viability Dye Red 780) as per the manufacturer’s instructions.
- Add surface-staining antibodies to the samples as per the manufacturer’s instructions. See protocol for FACS staining. For surface antigen staining, we recommend:
- Fluorochrome-conjugated Anti-Human CD3 Antibody, Clone UCHT1
- Fluorochrome-conjugated Anti-Human CD8 Antibody, Clone RPA-T8
- Fluorochrome-conjugated Anti-Human CD4, Clone RPA-T4
- Wash cells twice in D-PBS + 2% FBS or staining buffer of choice.
- Add Fixation Buffer to fix the cells and permeabilize using Intracellular Permeabilization Buffer, following the instructions on the Product Information Sheets. Add intracellular antibodies to the samples as per the manufacturer’s instructions, e.g.:
- Fluorochrome-conjugated Anti-Human IFN-gamma Antibody, Clone 1-D1K
- Fluorochrome-conjugated Anti-Human TNF-alpha Antibody, Clone MT15B15
- Wash cells and analyze by flow cytometry.
- Using a commercially available ELISpot kit, e.g. human IFN-gamma, prepare ELISpot plates as per the manufacturer’s instructions.
- Starting with the peptide pool stock(s), prepare 3X (e.g. 3 µg/mL per peptide) working solution(s) in the cell culture medium of choice.
- Add 50 µL of the 3X working solution to each well of the ELISpot plate.
- Add 100 µL of cell suspension (e.g. 2.5 x 105 PBMCs) to each well, pipetting gently down the side of the wells.
- Incubate the cells with peptide at 37°C, 5% CO2 for the desired incubation time. Ensure the plate is not disturbed during this time. Typically 18 - 48 hours is recommended but this should be optimized for your experiment and analyte of choice.
- After the incubation time is complete, continue to process the ELISpot as per the manufacturer’s instructions.
T Cell ELISpot
Expected Results: Antigen-Specific T Cell Responses Elicited by Peptide Pools
In this study, the presence of antigen-specific T cells was evaluated in human peripheral blood mononuclear cells (PBMCs). Two peptide pools were tested, which included (1) CEF (HLA Class I Control) Peptide Pool, frequently used as a positive control as it contains peptide sequences from three viruses that commonly infect humans (namely CMV, EBV & Influenza), and (2) SARS-CoV-2 Spike Protein Peptide Pool. Following peptide pool stimulation, we evaluated the number of antigen-specific T-cells that were activated by each peptide pool, utilizing flow cytometry to detect the frequency of IFN-gamma and TNF-alpha producing T cells (Figure 1), as well as ELISpot to quantify the number of IFN-gamma-secreting cells (Figure 2). The results presented are from a representative donor and show that CEF and SARS-CoV-2 Peptide Pools specifically induced activation of a small subset of T cells when compared to baseline (No Peptide), confirming the presence of CMV, EBV, and/or Influenza, and SARS-CoV-2 reactive T cells in this individual. It should be noted that T cell responses will vary between donors and tested antigens. These results successfully demonstrate the ability of peptide pools to induce antigen-specific T cell responses and validate two methods for detecting these cells.
Figure 1. Evaluation of Antigen-Specific T Cell Activation by Peptide Pools Using Intracellular Cytokine Staining of IFN-gamma and TNF-alpha
Peripheral Blood Mononuclear Cells (PBMCs) were cultured in ImmunoCult™-XF T Cell Expansion Medium supplemented with either CEF (HLA Class I Control) Peptide Pool, SARS-CoV-2 (Spike Protein) Peptide Pool, or left unstimulated (No Peptide) for 6 hours, in the presence of Brefeldin A. Cells were harvested and stained with a GloCell Fixable Viability dye, followed by anti-human CD3, CD8, and CD4 antibodies. Cells were then fixed and stained intracellularly for anti-human IFN-gamma and TNF-alpha. To detect the frequency of IFN-gamma and TNF-alpha producing T cells stimulated by peptide pools, cells were analyzed by flow cytometry and gated on viable CD3+CD8+CD4- (top row) and CD3+CD8-CD4+ (bottom row) cells. Data shown are from a single donor.
Figure 2. Evaluation of Antigen-Specific T Cell Activation by Peptide Pools Using an IFN-gamma ELISpot Assay
Peripheral Blood Mononuclear Cells (PBMCs) were cultured in ImmunoCult™-XF T Cell Expansion Medium supplemented with either CEF (HLA Class I Control) Peptide Pool, SARS-CoV-2 (Spike Protein) Peptide Pool, or left unstimulated (No Peptide) for 18 hours on commercially available pre-coated human IFN-gamma ELISpot plates. All wells were seeded at 2.5 x 105 cells/well. Following the incubation period, plates were processed as per the manufacturer's instructions and imaged on an automatic ELISpot reader. Each spot represents one antigen-specific T cell secreting IFN-gamma. When compared to peptide-free conditions (No Peptide), the number of T cells secreting IFN-gamma was increased in wells that were stimulated by CEF (HLA Class I Control) and SARS-CoV-2 (Spike Protein) Peptide Pools. Data shown are representative wells from a single donor.
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