Introduction

Dendritic cells (DCs) are powerful antigen-presenting cells (APCs) that process exogenous antigens and present them to CD4⁺ and CD8⁺ T cells to generate immune responses. The presentation of exogenous antigens on major histocompatibility complex (MHC) class I molecules to CD8⁺ T cells is known as cross-presentation and is essential to induce antigen-specific responses of CD8⁺ T cells to fight infections and disease. As such, co-culturing DCs and CD8⁺ T cells is a useful method for studying the mechanisms of antigen presentation as well as T cell activation and proliferation. T cells expanded from the co-culture system can be further used to study the presentation of tumor antigens, T cell-mediated cytotoxicity, and memory T cell formation. Results from such studies may be used to develop T cell-mediated cancer treatments or vaccines that induce long-lasting immune memory and protection.

This technical bulletin describes, in detail, how to set up DC and CD8⁺ T cell co-culture experiments that generate antigen-specific CD8⁺ T cells, as well as how to assess CD8⁺ T cell proliferation, functionality, and killing activity. The protocols within the technical bulletin have undergone in-house testing and validation, and provide a reliable, comprehensive guide to support your entire DC/T cell research workflow.

Protocols

The following protocols describe how to set up a co-culture with dendritic cells (DCs) and CD8⁺ T cells, as well as options for downstream functional analysis (see Figure 1). To study DC priming activity, the recommendation is to set up a short-term co-culture of 6 days. To study CD8⁺ T cell activation, proliferation, immune memory, and effects of re-exposure to the antigen, the recommendation is to set up a long-term, i.e. 10 - 12 days, co-culture system.

Isolate Human Monocytes and T Cells

1. Obtain peripheral blood mononuclear cells (PBMCs) from leukopaks (Catalog #70500)* or Human Whole Peripheral Blood (Catalog #70504)*. For more details, refer to our leukopak processing and PBMC isolation protocols, respectively.
2. Use one fraction of PBMCs to isolate monocytes and cryopreserve the remaining PBMCs to use at a later time for CD8⁺ T cell isolation and, if required, for antigen-presenting cell (APCs) enrichment. This will allow the establishment of an autologous co-culture system.
3. Isolate human CD14⁺CD16^- monocytes from PBMCs using EasySep™ Human Monocyte Isolation Kit (Catalog #19359). Refer to the Product Information Sheet for details.
4. When ready to start the co-culture system, thaw autologous cryopreserved PBMCs, culture overnight, and then isolate CD8⁺ T cells from the PBMCs using EasySep™ Human CD8⁺ T Cell Isolation Kit (Catalog #17953), following the instructions on the Product Information Sheet.

Note: PBMCs can be obtained from large-volume sources, including fresh leukopaks* or human whole blood*. Additionally, you can save time by using pre-isolated fresh or frozen PBMCs* for your experiments.

*Certain products are only available in select territories. Please contact your Sales Representative or the Product & Scientific Support team at techsupport@stemcell.com for further information.

Note: Dendritic cells and T cells used in the culture system can either be autologous (from the same donor) or allogeneic (derived from different donors). For co-cultures creating antigen-presenting conditions, it is recommended to use autologous cells. This means that the DCs are fully MHC-matched to the T cells because they come from the same donor, thereby maximizing the breadth of their antigen presentation to potentially reactive T cells.

Generate Mo-DCs and Pulse with Peptide Pool

Figure 2. Protocol Diagram: Generation and Differentiation of Mo-DCs

Generate mature Mo-DCs from isolated monocytes. For optimal cell yield in this application, we recommend using EasySep™ Human Monocyte Isolation Kit. Culture the cells in ImmunoCult™-ACF Dendritic Cell Medium with added ImmunoCult™-ACF Dendritic Cell Differentiation Supplement for three days at 37°C. On Day 3, remove the medium and add fresh medium with added differentiation supplement, and incubate at 37°C for 2 more days. At Day 5, without changing the medium, add ImmunoCult™ Dendritic Cell Maturation Supplement and desired peptide pool(s) to the culture. On Day 6, harvest fully mature DCs for downstream applications.

The steps below describe how to generate mature Mo-DCs using ImmunoCult™ Dendritic Cell Culture Kit (Catalog #10985) following the Product Information Sheet, with modifications to pulse Mo-DCs with a peptide pool on Day 5 of culture (see Figure 2). Refer to the ImmunoCult™ Dendritic Cell Culture Kit Product Information Sheet for details omitted in this summary. For a list of suggested materials and products used in this protocol, see Table 1.

1. On Day 0, seed monocytes at 1 x 10⁶ cells/mL in appropriate tissue cultureware.
2. On Day 3, replace the medium with fresh ImmunoCult™-ACF Dendritic Cell Medium (Catalog #10987).
3. On Day 5, add ImmunoCult™ Dendritic Cell Maturation Supplement (Catalog #10989) directly to the cultures at a 1 in 100 dilution. Add specific desired peptides (e.g. CMV (pp65) Peptide Pool (Catalog #100-0668), Influenza (HLA Class I Control) Peptide Pool (Catalog #100-0672), MART 1(26-35) (peptides&elephants GmbH, Catalog #EP09832_1), or CMV Sub Peptide Pool (peptides&elephants GmBH, Catalog #LB01713) at a concentration of 1 µg/mL per peptide).
4. On Day 6, harvest mature Mo-DCs by gently pipetting up and down to ensure all cells are in suspension, then transfer to an appropriate tube.
5. Wash the cells by adding an equivalent volume of ImmunoCult™-XF T Cell Expansion Medium (Catalog #10981) and gently pipetting up and down to make sure the cells are resuspended in the medium. Centrifuge at 300 x g for 10 minutes at room temperature. Remove and discard the supernatant.
6. Resuspend the cells in ImmunoCult™-XF T Cell Expansion Medium. Repeat step 5. After centrifugation, remove and discard the supernatant and resuspend the cells with ImmunoCult™-XF T Cell Expansion Medium at a concentration of 5 x 10⁵ cells/mL.

Note: Selecting Peptide Pools

A peptide pool is a mixture of short peptide fragments of a specific protein or antigen that can stimulate T cells with a specific T cell receptor (TCR) specificity. Peptide pools allow researchers to screen and identify T cells that can recognize and respond to specific antigens in a complex mixture, such as those found in pathogens or cancer cells. The peptide chosen will depend on the research interest and question being addressed. The peptide pools used here are presented by human leukocyte antigen (HLA) class I molecules on DCs and interact with TCRs on CD8⁺ T cells to induce activation and proliferation. The CMV (pp65) Peptide Pool, Influenza (HLA Class I Control) Peptide Pool, and MART 1 (26-35) peptides are often good positive controls, as most individuals will have circulating CD8⁺ T cells that recognize these antigens.

Mo-DC and T Cell Co-Cultures

The length for co-culture of Mo-DCs and CD8⁺ T cells may vary depending on the specific scientific questions being addressed. Short-term co-culture of 6 days (see Figure 3) is typically performed to study the activation of dendritic cells and their priming of CD8⁺ T cells. However, longer-term co-culture of 10 - 12 days (see Figure 4) will generate a pool of expanded antigen-specific CD8⁺ T cells that can be used in downstream functional assays, such as CD8⁺ T cell-mediated cytotoxicity assays. Optimal cell yields depend on maintenance of cell health, which in turn will depend on following the recommended schedule of feeding and medium changes.

Option 1: Short-Term Co-Culture

This protocol is designed to promote the activation and proliferation of antigen-specific CD8⁺ T cells for 6 days within a co-culture system with autologous antigen presenting Mo-DCs (see Figure 3). Optimal cell yields depend on maintenance of cell health, which largely depends on following the recommended schedule of feeding and medium changes. For a list of suggested materials and products used in this protocol, see Table 1.

1. Thaw autologous and cryopreserved PBMCs and culture overnight (to let the freshly thawed frozen cells rest) in ImmunoCult™-XF T Cell Expansion Medium, supplemented with 5 ng/mL Human Recombinant IL-7 (Catalog #78053).
2. On Day 0, isolate CD8⁺ T cells from PBMCs in step 1 using EasySep™ Human CD8⁺ T Cell Isolation Kit and label the cells with 5 μM of cell proliferation tracking dye CellTrace™ Violet Proliferation Dye (Thermo Fisher Scientific, Catalog #C34557), as per manufacturer’s instructions. Add quench solution with 10% FBS and then wash cells by adding an equivalent volume of ImmunoCult™-XF T cell Expansion Medium and centrifuging at 300 x g for 10 minutes at room temperature. Repeat the wash. Resuspend labeled CD8⁺ T cells at a concentration of 2 x 10⁶ cells/mL in ImmunoCult™-XF T Cell Expansion Medium.
3. Set up co-cultures by seeding 0.5 mL of the peptide-pulsed DC suspension (5 x 10⁵ cells/mL) with 0.5 mL of the CD8⁺ T cell suspension (2 x 10⁶ cells/mL) into each well of a 24-well tissue culture plate to obtain a 1:4 ratio of Mo-DCs to CD8⁺ T cells.
4. On Day 5 or 6, harvest the co-culture by gently pipetting up and down to ensure all cells are in suspension, then transfer to an appropriate centrifuge tube.
5. Wash cells by adding an equivalent volume of ImmunoCult™-XF T Cell Expansion Medium and centrifuge at 300 x g for 10 minutes at room temperature. Remove and discard the supernatant.
6. Repeat step 5 by resuspending cells in medium and centrifuge at room temperature. Remove the supernatant and resuspend the cell pellet in ImmunoCult™-XF T Cell Expansion Medium.
7. Determine the cell concentration and dilute by adding medium to obtain the desired final cell concentration for downstream applications and analysis.
8. Cells are ready for downstream analyses, including proliferation and phenotypic analyses, and quantification of antigen-specific T cells using tetramer staining.

For phenotypic analysis, the recommendation is to stain the cells with corresponding HLA class I tetramers, and fluorescent antibodies specific for human CD8 (clone RPA-T8), CD3 (clone SK7), CD25 (clone BC96), and CD279 (PD1; clone EH12.2H7), and viability dye DRAQ7™ (BioLegend, Catalog #424001). Stain the cells following the recommended protocol as per manufacturer's instructions, and analyze using flow cytometry (see Figure 5). To quantify the number of antigen-specific CD8⁺ T cells, multiply the frequency of viable tetramer CD8⁺ T cells by the cell concentration determined in step 7.

Note: Tetramer staining is a powerful technique for analyzing antigen-specific T cells due to its high specificity and sensitivity in identifying and quantifying these cells and allowing researchers to study the immune response to specific antigens. The tetramers are bioengineered with four MHC-peptide molecules and provide stable binding to T cells that recognize a specific antigen. By labeling tetramers with fluorescent dyes, e.g. phycoerythrin (PE), researchers can use flow cytometry to identify and analyze antigen-specific T cell functionality in a sample, which allows accurate and precise quantification of antigen-specific T cells. The tetramer is selected depending on the HLA alleles of the donor and the specific peptide used to activate the cells, e.g. if the cells are stimulated with CMV (pp65) Peptide Pool, then stain with iTAg Tetramer/PE – HLA-A*02:01 CMV pp65 (NLVPMVATV) (MBL International Corporation, Catalog #TB-0010-1; see experiment data shown in Figure 6); if the cells are stimulated with MART 1(26-35) peptides, then stain with iTAg Tetramer/PE – HLA-A*02:01 Mart-1 (ELAGIGILTV) (MBL International Corporation, Catalog #TB-0009-1).

Option 2: Long-Term Co-Culture

This protocol is designed to promote the proliferation/activation of antigen-specific CD8⁺ T cells for 10 - 14 days within a co-culture system with autologous antigen-presenting Mo-DCs (Figure 4). Optimal cell yields depend on maintenance of cell health, which largely depends on following the recommended schedule of feeding and medium changes. For a list of suggested materials and products used in this protocol, see Table 1.

1. Thaw autologous and cryopreserved PBMCs and culture overnight in ImmunoCult™-XF T Cell Expansion Medium, supplemented with 5 ng/mL Human Recombinant IL-7.
2. On Day 0, isolate CD8⁺ T cells from PBMCs in step 1 using EasySep™ Human CD8⁺ T Cell Isolation Kit. Resuspend isolated cells at a concentration of 2 x 10⁶ cells/mL in ImmunoCult™-XF T Cell Expansion Medium supplemented with 60 ng/mL of Human Recombinant IL-21 (Catalog #78193).
3. Set up co-cultures by seeding 0.5 mL of the peptide-pulsed dendritic cell suspension (5 x 10⁵ cells/mL) with 0.5 mL of the CD8⁺ T cell suspension (2 x 10⁶ cells/mL) into each well of a 24-well tissue culture plate to obtain a 1:4 ratio of Mo-DCs to CD8⁺ T cells.
4. On Day 3, add another 1 mL of fresh ImmunoCult™-XF T Cell Expansion Medium with Human Recombinant IL-7 and Human Recombinant IL-15 (Catalog #78031) to each well, to bring the final IL-7 and IL-15 concentrations to 5 ng/mL for each cytokine.
5. On Day 5, transfer the contents of each co-culture well from the 24-well tissue culture plate to a 6-well tissue culture plate. Add an additional 2 mL of fresh medium, supplemented with 5 ng/mL of IL-7 and 5 ng/mL of IL-15, to top up the existing 2 mL volume of cell culture.
6. On Day 7, split each co-culture well into 2 wells by removing 2 mL of cell suspension from the 6-well tissue culture plates and seed the cells into a fresh well of a 6-well tissue culture plate. Add an additional 2 mL of fresh medium, supplemented with 10 ng/mL of IL-7 and 10 ng/mL of IL-15, to each well and culture the cells for another 3 days.
7. On Day 10, harvest the cells by gently pipetting up and down to ensure all cells are in suspension, and transfer the cells to an appropriate tube for downstream analysis, functional assays, or to be seeded onto tissue culture plates for further expansion.

Note: By Day 10 of the co-culture, nearly all of the cells constitute expanded T cells (see Figure 6) with very few Mo-DCs still present in the culture.

See below for protocols and data examples to:

• Quantify antigen-specific T cells with tetramer staining (see Figure 6)
• Assess degranulation (CD107) and production of interferon-γ (IFN-γ) and CD137 expression (Figure 7)
• Isolate antigen-specific T cells (Figure 8A)
• Assess T cell killing activity (Figure 8B)

Assess T Cell Functions (Degranulation, IFN-γ Production, and 4-1BB (CD137) Expression)

T cell activation can be measured by detecting degranulation, interferon-γ (IFN-γ) production, and 4-1BB (CD137) expression, which provide valuable insights into the immune response and can help researchers understand the efficacy of potential immunotherapies. Measuring CD107 surface expression is a common method to quantify degranulation. When activated by a specific antigen, CD8⁺ T cells begin to degranulate, transport CD107 to the cell surface, and deliver cytotoxic proteins to target cells (Betts et al., 2003). The exposure of antigens to CD8⁺ T cells also induces the production of IFN-γ that can be measured by intracellular cytokine staining. The surface glycoprotein CD137 is also induced by antigen exposure and is a potent co-stimulatory signal that promotes T cell proliferation and formation of memory cells, enhances survival, and increases the production of IFN-γ (Fröhlich et al., 2020). For a list of suggested materials and products used in this protocol, see Table 1.

Part 1: Set Up Antigen-Presenting Cells for CD8⁺ T Cell Recall Immune Responses

The DC/CD8⁺ T cell co-culture is required to expand antigen-specific CD8⁺ T cells. After the first 3 or 4 days of Mo-DC and CD8⁺ T cell co-culture, most of the T cell expansion is driven by cytokines (IL-15 and IL-7). Therefore, T cells need to be reactivated with an autologous source of antigen-presenting cells (APCs) and fresh peptides to create a memory-like recall response to antigen, and to induce detectable levels of IFN-γ production and other activation markers. The steps below describe how to set up APCs to add to the expanded CD8⁺ T cells to measure cytokine production.

1. On Day 10, thaw autologous PBMCs to use as a source of APCs for CD8⁺ T cell reactivation.
2. Enrich APCs from autologous PBMCs by depleting CD3⁺ and CD2⁺ cells to remove T cells and NK cells. We recommend using EasySep™ Human CD3 Positive Selection Kit II (Catalog #17851) and EasySep™ Human CD2 Positive Selection Kit II (Catalog # 17833) with the purple EasySep™ Magnet (Catalog #18000) as described below:
   
   1. Dilute PBMCs to 5 x 10⁷ cells/mL and transfer to a 5 mL polystyrene round-bottom tube.
   2. Add EasySep™ Human CD3 Positive Selection Cocktail II and EasySep™ Human CD2 Positive Selection Cocktail at 150 μL/mL each, and incubate for 5 minutes.
   3. Add 150 μL/mL EasySep™ Dextran RapidSpheres™ (Component #50100) to the sample, mix, and incubate at room temperature for 5 minutes.
   4. Add EasySep™ Buffer (Catalog #20144) to top up to 2.5 mL, place the tube into the purple EasySep™ Magnet, and incubate at room temperature for 5 minutes.
   5. Pick up the magnet and, in one continuous motion, invert the magnet and tube, pouring off the supernatant to a new 5 mL polystyrene round-bottom tube. This new tube contains the enriched cells.
   6. Add 75 μL/mL EasySep™ Dextran RapidSpheres™ (Component #50100) to the enriched fraction collected from the pour-off. Mix and incubate at room temperature for 1 minute.
   7. Place the tube into the purple EasySep™ Magnet and incubate for 5 minutes.
   8. Pick up the magnet, and in one continuous motion, invert the magnet and tube, pouring off the supernatant to a new 5 mL polystyrene round-bottom tube.
   9. Once again, place the tube into the purple EasySep™ Magnet and incubate for another 5 minutes.
   10. Pick up the magnet, and in one continuous motion, invert the magnet and tube, pouring off the supernatant to a new 5 mL polystyrene round-bottom tube. The pour-off fraction will contain the enriched APCs.
3. Centrifuge the enriched APCs at 300 x g for 10 minutes. Resuspend in 2.5 mL phosphate-buffered saline (PBS) and centrifuge at 300 x g for 10 minutes.
4. Remove the supernatant and resuspend the cells in 1 mL PBS supplemented with cell proliferation tracking dye (5 μM of CellTrace™ Violet Proliferation Dye; Thermo Fisher Scientific, Catalog #34557), as per manufacturer’s instructions. This step will help distinguish the APCs from the responder T cells when analyzing the cells by flow cytometry. Add a quench solution with 10% FBS and then wash cells by adding an equivalent volume of ImmunoCult™-XF T cell Expansion Medium and centrifuging at 300 x g for 10 minutes.
5. Resuspend fluorescently labeled APCs in 1 mL ImmunoCult™-XF T Cell Expansion Medium.
6. Pulse the APCs in the 5 mL polystyrene round-bottom tube used for APC enrichment with the same peptide pool that was previously used to activate Mo-DCs in the Mo-DCs maturation step, at 1 μg/mL per peptide(s). Leave a fraction of the APCs untreated as a negative control. Incubate the APCs with peptide at 37°C for 2 -16 hours. Add 2 mL of ImmunoCult™-XF T Cell Expansion Medium to the APCs and centrifuge APCs at 300 x g for 10 minutes to remove excess peptides.
7. Decant the supernatant, resuspend APCs in 3 mL ImmunoCult™-XF T Cell Expansion Medium, and centrifuge at 300 x g for 10 minutes. Decant the supernatant again, resuspend the APCs in ImmunoCult™-XF T Cell Expansion Medium, and dilute the cells to 1 x 10⁵ cells/mL. The APCs are now ready to be used to assess CD8⁺ T cell degranulation and IFN-γ production, described below.

Part 2: Assess Degranulation and IFN-γ Production (Following 10 Days of Co-Culture and CD8⁺ T Cell Expansion)

1. Dilute expanded CD8⁺ T cells (from “Long-Term Co-Culture” protocol) to 1 x 10⁵ cells/mL. Add 100 μL of expanded CD8⁺ T cells to 100 μL of peptide-pulsed and fluorescently labeled APCs (at 1 x 10⁵ cells/mL; from “Set-Up of Antigen-Presenting Cells for CD8⁺ T Cell Recall Immune Responses”) in a round-bottom 96-well tissue culture plate and incubate at 37°C. The final concentration for each cell type should be 1 x 10⁴ cells/well.
2. Add PE anti-human CD107a (LAMP-1) antibody (clone H4A3; Biolgend, Catalog #328607) to APC/CD8⁺ T cell co-culture at 0.2 μL/well. Incubate for 1 hour.
3. Add BD GolgiStop™ protein transport inhibitor (Monensin) (BD Biosciences, Catalog #554724) to the co-culture, as per the manufacturer’s instructions.
4. Incubate for 5 hours.
5. Harvest the cells from the plates to analyze by flow cytometry.