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. Author manuscript; available in PMC: 2026 Feb 3.
Published in final edited form as: Methods Cell Biol. 2025 Apr 9;201:109–120. doi: 10.1016/bs.mcb.2025.03.009

Peripheral blood mononuclear cell (PBMC)- based functional evaluation of human T cell response to suppressive cells and immune-oncology therapeutics

Mahmoud S Alghamri 1,2, Brandon L McClellan 1,2, Kaushik Banerjee 1,2, Jorge A Peña Agudelo 1,2, Pedro R Lowenstein 1,2, Maria G Castro 1,2
PMCID: PMC12862213  NIHMSID: NIHMS2047042  PMID: 41620272

Abstract

Cancer immunotherapies leverage the immune response to target cancer cells with T cells playing a pivotal role. However, tumor microenvironments often harbor immune suppressive elements hindering T cell function. This chapter describes in vitro T cell stimulation assays, analyzing proliferation, inhibitory marker expression, and effector functions to assess the impact of immune suppression on T cell responses. These assays also evaluate the efficacy of immunotherapeutic interventions in overcoming immune suppression and enhancing anti-tumor immunity, thereby unraveling the intricate T cell-tumor microenvironment dynamics for more effective cancer immunotherapies.

Keywords: T cell activation, Immuno-Oncology, Inhibitory cells, Immunotherapy

6. SUMMARY

Cancer immunotherapies aim to enhance immune responses to fight cancer, where cytotoxic T cells play a pivotal in targeting tumors. Once activated, CD8 T cells can kill tumors either by secreting cytokines like IFN-γ or through the release of granzymes and perforins, while CD4 T-helper cells support strong immune responses through pro- inflammatory cytokines. However, the tumor microenvironment often harbors cells such as Tregs, macrophages, and MDSCs, which inhibit anti-tumor immunity. Therefore, it is important to elucidate the efficacy of immunotherapeutic interventions and unravel the intricate dynamics of T cells in the tumor microenvironment for more effective cancer immunotherapies under varied conditions. This chapter outlines the process for isolating PBMCs and CD8 T cells from leukopaks, proliferation labelling CD8 T cells, and activating them under different conditions followed by functional studies. The steps start with diluting blood with DPBS, layering it over Ficoll, and centrifuging to collect the buffy coat. CD8 T cells are then isolated, labeled with CFSE, and activated with Dynabeads for proliferation assays. With this chapter users may try potential therapies or add other cells on CD8 T cells analyze the effect on T cell function. This would provide insight as to the effects of the co- cultured cell or therapeutic on CD8 T cells. These protocols are crucial for understanding how different molecules and cell interactions affect T cell mediated anti-tumor responses.

1. INTRODUCTION

Cancer immunotherapies are treatment strategies geared to promote the immune response within the body to treat cancer. T cells, especially cytotoxic T cells, are a critical component in the fight against cancer and are the focus of many cancer immunotherapies to improve the anti- tumor immune response (Oh et al., 2021; Waldman et al., 2020). T cells become activated via three required signaling mechanisms. The signals include: (1) the recognition and binding of the T cell receptor (TCR) to its cognate antigen presented on a major histocompatibility complex (MHC), (2) the binding of T cell surface protein CD28 to B7 proteins (CD80 and CD86) on the surface of antigen presenting cells (APCs), and (3) the cytokine-mediated signaling cascade that supports effector function and drives differentiation of activated T cells into T cell subtypes (Hwang et al., 2020; Lee et al., 2020). Upon activation, the cells carry out their effector functions depending on the type of T cell. CD8 T cells are effector cells which mediate tumor cell killing directly or indirectly. CD8 T cells release granules of perforin and granzymes which cause tumor cell death directly (Waldman et al., 2020). CD8 T cells trigger tumor cell killing indirectly by secreting cytokines such as interferon-gamma (IFN-γ) and tumor necrosis factor-alpha (TNF-α) thereby promoting inflammation and tumor cell apoptosis (de Visser & Joyce, 2023; Waldman et al., 2020). Helper CD4 T (Th) cells are notable for their contribution to tumor cell killing by assisting in the development of robust and durable immune responses via the release of proinflammatory cytokines and chemokines (de Visser & Joyce, 2023).

In addition to anti-tumor immune cells, tumor microenvironments host several forms of immune suppression. Immunosuppressive cells such as cancer cells, tumor-associated macrophages, myeloid derived suppressor cells (MDSCs), and CD4 T regulatory (Treg) cells work to suppress anti-tumor immunity (de Visser & Joyce, 2023; Tie et al., 2022). They secrete molecules such as, transforming growth factor-beta (TGFβ), interleukin-10 (IL-10), prostaglandin E2 (PGE2), reactive oxygen species, and they express inhibitory proteins including the immune checkpoint receptors (de Visser & Joyce, 2023; Tie et al., 2022). These features of the tumor microenvironment help enable tumor cells to evade T cells (Tie et al., 2022).

This chapter details in vitro assays for evaluating T cell responses to chosen molecules or cell interactions. Using human peripheral blood mononuclear cells, the T cells can be stimulated under varied conditions and analyzed for the effect on several T cell characteristics. We describe: (1) the protocol of T cell stimulation with the optional addition of potential therapeutics (2) the staining method to measure T cell proliferation using the fluorescent cell staining dye carboxyfluorescein succinimidyl ester (CFSE), (3) coculturing T cells with other cells, (4) the analysis of the T cells to measure the effects of therapies and cocultured cells on T cell function. We believe that this is particularly important in the assessment of isolated effects of various molecules and cells on the ability of T cells to carry out their anti-tumor properties.

2. MATERIALS

2.1. REAGENTS

Below are reagents that are regularly used for this protocol, but there are various alternatives commercially available. To make this protocol more accessible, we provide a list of alternative reagents and equipment. (see note 1)

  • Leukopak (STEMCELL Technologies; 70500)

  • DPBS, no calcium, no magnesium (Gibco; 14190250)

  • Cytiva Ficoll-Paque PREMIUM (Ficoll; Thermo Fisher Scientific; 45–001-751)

  • ACK lysis buffer (Gibco; A1049201)

  • Cryostor CS10 freezing media (STEMCELL Technologies; 100–1061)

  • CD8 T cell isolation kit (Miltenyi Biotec; 130–096-495)
    • Biotin-Antibody Cocktail (in included in kit)
    • CD8+ T cell MicroBead cocktail (included in kit)
  • CD8 T cell isolation kit buffer
    • MACS BSA stock solution (Miltenyi Biotec; 130–091-376)
    • AutoMACS rinsing solution (Miltenyi Biotec; 130–091-222)

  • RPMI 1640 Medium (Gibco; 11875119)

  • Fetal Bovine Serum (FBS; Heat-inactivated before use; Gibco; A5670701)

  • 2-Mercaptoethanol (BME; Gibco; 21985023)

  • Penicillin-Streptomycin (Pen-Strep; Gibco; 15140122)

  • Carboxyfluorescein succinimidyl ester (CFSE; Invitrogen; 50–169-50)

  • Dimethyl sulfoxide (DMSO; Thermo Scientific Chemicals; J66650.AE)

  • Dynabead Human T-Activator CD3/CD28 (Gibco; 11131D)

  • Human IL-2 Recombinant Protein (rIL-2; Gibco; PHC0026)

  • GolgiPlug Protein Transport Inhibitor (Containing Brefeldin A) (BD; 555029)

  • GolgiStop Protein Transport Inhibitor (Containing Monensin) (BD; 554724)

  • Propidium Iodide (PI; Invitrogen; P1304MP)

2.2. EQUIPMENT

The equipment below is necessary for the completion of the protocol. An example in parenthesis accompanies each equipment but a similar functioning alternative may be used.

  • Dynabeads magnet (DynaMag-2 Magnet; Invitrogen; 12321D)

  • MACS separator (QuadroMACS Separator; Miltenyi Biotec; 130–090-976)

  • LS columns (Miltenyi Biotec; 130–042-401)

  • Hemacytometer (Hausser Scientific Bright Line Counting Chamber; Thermo Fisher Scientific, 02–671-51B)

  • Microscope (Olympus Stereomicroscope System SZX16; Olympus)

  • Centrifuge (Allegra 6R Refrigerated Benchtop Centrifuge, Beckman Coulter)

  • Flow cytometer (BD FACSAria II flow cytometer; BD Biosciences)

  • Humidified CO2 incubator (Fisherbrand Isotemp Water Jacketed CO2 Incubator, 184 L, Stainless Steel; Thermo Fisher Scientific; 11–676-600)

3. MR. FROSTY FREEZING CONTAINER (THERMO SCIENTIFIC; 5100–0036)T CELL ISOLATION FROM LEUKOPAKS

3.1. ISOLATION OF PBMCS FROM LEUKOPAKS:

  1. All steps should be performed in Class II biological safety cabinets (see note 2).

  2. Cut the tip of the tube that is coming out of the leukopak.

  3. Pour 100 mL of blood into a 500 mL glass beaker.

  4. To each 100 mL of blood, add 100 mL of DPBS.

  5. For 200 mL of leukopak blood, label 12 tubes (50 mL) and add 13 mL of Ficoll to the bottom of the tubes.

  6. Carefully layer 15 mL of DPBS diluted blood (dropwise) to the 13 mL of Ficoll in each tube.

  7. Make sure that two layers formed.

  8. Centrifuge the tubes at 800 x g for 30 min at 20 oC. Ensure the centrifuge decelerate with brakes off. (Weigh tubes to make sure balanced)

  9. Label four new 50 mL tubes for the buffy coats.

  10. After centrifugation is completed, collect buffy coat from three tubes into each of the four pre- labelled tubes. Approximately 3–5 mLs of buffy coat collected/tube.

  11. Bring up the buffy coat tube volume to 50 mL with DPBS.

  12. Centrifuge the tubes at 800 x g for 10 min at 20 oC (see note 3).

  13. Aspirate and discard the supernatant.

  14. Add 1 mL of ACK Lysis buffer to the pellet and resuspend the cells.

  15. Add additional 8 mL of ACK buffer to the cells.

  16. Leave the tube at room temperature (RT) for 3–5 min.

  17. At the end of the incubation, add 30 mL of DPBS to the tube.

  18. Centrifuge the tube at 300 x g for 10 min at 20 oC.

  19. Aspirate and discard the supernatant.

  20. Resuspend the cell pellets of PBMCs in 1 mL of DPBS.

  21. Combine all tubes for each donor.

  22. Count the PBMCs. For counting, the hemacytometer may be used.

  23. Centrifuge the cells at 300 x g for 10 min.

  24. Aspirate and discard the supernatant.

  25. Resuspend the pellet in Cryostor CS10 freezing media.

  26. Freeze the PBMCs in a Mr. Frosty freezing container for a controlled rate freeze about −1°C/min until −80 °C. (see note 4)

  27. Transfer the PBMC vials to liquid nitrogen for long-term storage.

3.2. ISOLATION OF CD8 T CELLS FROM PBMCS

CD8 T cell isolation was performed using a CD8 T cell isolation kit and LS column following the vendor recommendation with some modifications as follow.

  1. Prepare single cell suspension of PBMCs and determine the cell number, centrifuge them at 300 x g.

  2. Resuspend the pellet with 40 µL of buffer per 107 of total PBMCs.

  3. Add 10 µL of preprepared Biotin-Antibody Cocktail per 107 of total PBMCs.

  4. Incubate for 5 min in the refrigerator (2−8 °C). with continuous mixing

  5. After incubation, add 30 µL of buffer per 10⁷ total cells

  6. Add 20 µL of CD8 T Cell MicroBead Cocktail per 10⁷ total cells.

  7. Incubate for 10 min in the refrigerator (2−8 °C) with continuous mixing.

  8. Prepare a LS column by placing it in suitable MACS separator and rinsing it with 3 mL of buffer (see note 5).

  9. Apply cell suspension onto the column. Collect flow-through containing unlabeled cells, representing the enriched CD8 T cells (see note 6).

  10. Rinse column by adding 3 mL of buffer into it, and collecting unlabeled cells that pass through, representing the enriched CD8 T cells.

  11. Combine the collected solutions of cells that passed through the column, as these should be the CD8 T cells.

  12. Check the CD8 T cell enrichment by flow cytometry. If the yield <90% CD8 T cells, proceed with another round of CD8 T cell enrichment (see note 7).

4. ACTIVATING AND CULTURING CD8 T CELLS

4.1. LABELLING CD8 T CELLS

To measure cell proliferation, CFSE is used to fluorescently label the cells. As the cells proliferate, the dye expression on each cell is divided between the daughter cells resulting in successive, measurable waves of proliferation (Lyons et al., 2013).

  1. Prepare T cell media by adding 10% FBS, 55 µM BME, 100 units/mL Pen-Strep to the media base of RPMI 1640.

  2. After CD8 T cell isolation, centrifuge the cells at 500 x g, discard the supernatant and resuspend the cells in 1 mL of T cell media.

  3. Count the cells and confirm the desired number of cells.

  4. Separate cells for a CFSE negative control sample, as desired.

  5. Centrifuge the cells at 500 x g for 5 min at 4 °C.

  6. Prepare 10 mM stock solution of CFSE by adding 90 µL DMSO to a vial of CFSE.

  7. Dilute the stock to a working solution of 5 µM CFSE by transferring 2.5 µL of 10 mM CFSE to 37 °C pre-warmed 5 mL DPBS.

  8. For the samples with CFSE, resuspend the cells in 5 mL of 5 µM CFSE in a 50 mL conical tube.

  9. For the negative control, resuspend the no CFSE cells in 5 mL 37 °C pre-warmed DPBS in a 50 mL conical tube.

  10. Incubate the cells in the dark for 5 min in 37 °C water bath.

  11. At the end of the incubation, immediately add 20 mL of 37 °C pre-warmed T cell media to each tube.

  12. Centrifuge these 2 tubes at 500 x g for 5 min (see note 8)

  13. Resuspend the pellets in 10 mL of T cell media.

  14. Incubate the cells for 20 min to fully quench the CFSE (see note 9)

  15. Centrifuge the tubes 500 x g for 5 min and proceed to section 4.2.

4.2. CD8 T CELL ACTIVATION and CO-CULTURING

T cell activation was performed using Dynabead Human T-Activator CD3/CD28 following the manufacturer recommendation with some modifications.

  1. Resuspend the Dynabeads in the original vial (vortex for >30 sec, or tilt and rotate for 5 min).

  2. Transfer the desired volume of Dynabeads to a 1.5 mL tube.

  3. Add an equal volume of buffer, or at least 1 mL, and mix (vortex for 5 sec, or keep on a roller for at least 5 min).

  4. Place the tube on a magnet for 1 min and discard the supernatant.

  5. Remove the tube from the magnet and resuspend to wash the Dynabeads in the same volume of culture medium as the initial volume of Dynabeads taken from the vial in step 2.

  6. Count the purified T cells from section 4.1.

  7. Resuspend the T cells to achieve a concentration of 8 × 104 T cells per 100 µL of T cell media.

  8. Add 100 µL of T cell suspension into the desired wells of a round-bottomed 96-well plate.

  9. Prepare T cell media with rIL-2 (20 ng/mL) (note 10-11).

  10. Add 100 µL of rIL-2 solution to the 100 µL T cell suspension wells.

  11. Add 2 µL pre-washed and resuspended Dynabeads to obtain a bead-to-cell ratio of 1:1 (see note 12).

  12. Culture the CD8 T cells in varied conditions depending on the downstream readout.
    1. To test the effects of immune-oncology therapeutics, add the therapeutic agent to the T cell suspension wells.
    2. To evaluate the inhibitory potential of immune-modulatory cell populations, i.e. MDSCs, macrophages, etc., add different ratios of thesecells to T cells as previously described (Alghamri et al., 2020).

  13. For tumor cell killing assays, co-culture T cells with tumor cells at different ratios. The assay can also be performed in parallel with the addition of a therapeutic agent to assess the agent’s impact on CD8 T cell-mediated tumor cell killing.Incubate in a humidified CO2 incubator at 37 °C, for 4 days (see note 13).

  14. If cytokine production will be measured by intracellular staining, add brefeldin A or monensin 4 hours before collecting the cells to block the release of cytokines (see note 14).

  15. Centrifuge the plate of cells 300 x g for 5 min.

  16. Evaluate T cell function by flow cytometry (see note 15). (Fig 1)
    1. Use cell identification markers if performing co-culture assays to clearly distinguish the CD8 T cells from the cocultured cells.
    2. Measure expression of activation (CD25, CD69, CD44), functional markers (IFN-γ, granzyme B, perforin, TNF-α), proliferation (CFSE), dysfunctional T cell immune checkpoint markers (PD1, TIM3, LAG, CTLA-4), cell viability (PI).
    3. For tumor cell killing assays, use cell identification markers and cell viability dye (PI).

Fig 1.

Fig 1.

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Schematic illustrating the process isolation CD8 T cells from healthy human donor and performing an in vitro T cell proliferation and activation assay. (A) The schematic shows the isolation of human CD8 T cells directly from healthy human blood sample. First PBMCs are isolated from whole blood by Ficoll gradient. CD8 cells are isolated from PBMCs by anti-CD8 microbead labelling and magnetic column separation. These cells are further confirmed through flow cytometry. (B) Carboxyfluorescein succinimidyl ester (CFSE) labeled human CD8 T cells are co-cultured with the desired cells. In this example, the cocultured cells are CD11b+HLA-DR- CD14+ (monocytic) or CD11b+HLA-DR-CD15+ (polymorphonuclear) MDSCs sorted from tumor. The T cells are activated with Dynabeads and cultured for 4 days. As T cells divide in response to Dynabead stimulation, fluorescence intensity decreases. The effect of the cocultured cells on T cell proliferation and activation can be assessed by measuring the corresponding fluorescence intensity of the dye and CD8 T cell functional markers compared to the control population via flow cytometry.

Acknowledgements:

This work was supported by NIH/NINDS Grants, R37-NS094804, R01-NS105556, R01-NS122165, R21-NS123879–01 and 1R21NS107894 to M.G.C.; NIH/NINDS Grants R01-NS076991, R01NS122234, R01-NS122378, and R01-NS096756 to P.R.L.; NIH/NIBIB: R01-EB022563; NIH/NCI U01CA224160, 2P30CA46592; the Department of Neurosurgery, the Rogel Cancer Center, Program in Cancer Hematopoiesis and Immunology (CHI), the ChadTough Foundation, The Pediatric Brain Tumor Foundation, Ian’s Friends Foundation, Smiles for Sophie Forever Foundation, Leah’s Happy Hearts, the BioInnovations in Brain Cancer (BIBC), University of Michigan Biosciences Initiative to M.G.C. and P.R.L. RNA Biomedicine Grant F046166 to M.G.C.; NIH/NCI T32-CA009676 to M.A.

Footnotes

1.

List of alternative reagents and equipment. Leukopaks (patient-derived whole blood).

DPBS (HBSS; Gibco; 14170120). Cryostor CS10 freezing media (Recovery Cell Culture Freezing Medium, Gibco, 12648010). Mr. Frosty Freezing Container (any cell freezing container usually filled with isopropanol for freeze rate moderation; transferring the vial to a −20 °C freezer for 1 hour then the −80 °C freezer overnight is an alternative to the cell freezing container, yet this could result in decreased viability). CD8 T cell isolation kit buffer (PBS, pH 7.2, 0.5% BSA, 2 mM EDTA). CFSE (CellTrace dyes which come with a choice of fluorescent labels, e.g. CellTrace Violet; Invitrogen; C34557). Dynabead Human T-Activator CD3/CD28 (purified anti-CD3 and anti-CD28 antibodies; Invitrogen; 16–0037- 81, 16–0289-81). propidium iodide (7AAD; Invitrogen; A1310, or any live-dead stain).

2.

Cells must be kept sterile to prevent contamination.

3.

If the media seems cloudy with no pellet, do a second centrifugation at 800 x g for 10 min.

4.

If the cells are frozen, thaw PBMCs quickly in a 37 °C water bath. Dilute the thawed cells using the T cell medium using at least 5 times the frozen solution volume. Centrifuge at 300 x g for 5 min. Remove and discard the supernatant. Add T cell media and recount the cells.

5.

Always wait until the column reservoir is empty before proceeding to the next step.

6.

For magnetic separation of CD8 T cells, a minimum of 500 µL is required for magnetic separation. If necessary, add buffer to the cell suspension prior to adding through column.

7.

In case a higher CD8 T cell enrichment is required, it is recommended to do another round of CD8 T cell isolation by passing the cells into another LS column.

8.

The cell pellet should appear yellowish after the CFSE incubation.

9.

This step is critical, as CFSE is toxic to cells if not fully hydrolyzed.

10.

Skip the addition of rIL-2 if the cytokine interferes with the planned experiment, but omission of rIL-2 may lower T cell activation, viability, and proliferation.

11.

In addition or instead of rIL-2, other cytokines may be added to this step in order to differently stimulate or differentiate the T cells depending on the desired downstream experiment.

12.

The bead to T cell ratio determines the resultant activation state of the cultured T cells. For example, if the goal is to activate T cells, the recommended ratio is 1:1, however, to generate T cell exhaustion, we recommend using 5:1 bead to cells ratio.

13.

The duration of the incubation should be adjusted if studying chronic T cell stimulation.

For example, for studying chronic T cell stimulation-induced T cell exhaustion 6–10 days may be optimal.

14.

Although brefeldin A and monensin are used interchangeably to inhibit protein transport for secretory protein production analysis, there are reports of the certain cytokine levels being affected using one inhibitor instead of the other. As such, consider reviewing the literature when choosing brefeldin A or monensin for the protein of interest

15.

For flow cytometry applications, remove the beads prior to staining. Place the tube on a magnet for 1–2 min to separate the beads from the solution. Transfer the supernatant containing the cells to a new tube.

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