Method Article

Studying Interactions between Myeloid Cells and CAR T Cells In Vitro and In Vivo

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DOI:

10.3791/68013

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July 25th, 2025

 ,  ,  ,  ,  , 
1T Cell Engineering, Mayo Clinic, 2Department of Molecular Medicine, Mayo Clinic, 3Mayo Clinic Graduate School of Biomedical Sciences, 4Division of Hematology, Mayo Clinic, 5Department of Immunology, Mayo Clinic

Corresponding Authors: Saad S. Kenderian <kenderian.saad@mayo.edu>

In This Article

Summary

Given the limited xenograft models available to study interactions between human CART and myeloid cells, we established in vitro and in vivo models to understand the impacts of human macrophages on CART cells. Findings can potentially be generalized to evaluate macrophage roles in the tumor microenvironment and test macrophage-targeted immunotherapies.

Abstract

Chimeric antigen receptor T (CART) cell therapy has led to remarkable clinical successes treating hematological malignancies. However, most patients relapse within 1-2 years. Several mechanisms leading to CART resistance have been identified, including T cell inhibition by immunosuppressive myeloid cells. Growing evidence has shown that monocytes and macrophages in the tumor microenvironment (TME) contribute to poor CART performance and clinical outcomes. Preclinical models to study interactions between human macrophages and CART cells are limited. Here, we discuss approaches to understand the impacts from human macrophages on CART cells both in vitro and in vivo. We describe in vitro coculture models to show that immunosuppressive macrophages inhibit CART functions. Additionally, we report a xenograft model where human macrophages and tumor cells are subcutaneously engrafted in NSG mice to study M2-like macrophage interactions with CART cells and tumor cells. These models can be used to evaluate roles of macrophages in the TME and test macrophage-targeted cancer immunotherapies.

Introduction

Autologous chimeric antigen receptor T (CART) cell therapy has achieved clinical successes in treating relapsed or refractory hematological malignancies, which has led to US FDA approval of six CART products targeting CD19 or B cell maturation antigen (BCMA)1. Despite the impressive initial activity of CART cell therapy, most patients relapse within the first 1-2 years following treatment2. Several mechanisms of CART resistance have been identified, including suboptimal T cell fitness, antigen escape, and poor CART persistence, as well as inhibition mediated by the immunosuppressive tumor microenvironment (TME)2,3. Myeloid cells, such as monocytes and macrophages, often comprise a significant part of the TME and have been demonstrated to be major inhibitors of other immune cells, including T cells4,5. The detrimental effects of myeloid cells within the context of CART cell therapeutic efficacy have been reported in preclinical models and in clinical trials of both hematological and solid tumors3,6. Myeloid-mediated inhibition can occur from the beginning of the CART manufacturing process -- it has been demonstrated that monocytes in the starting apheresis product inhibit ex vivo CART expansion7,8. Given the accumulating evidence of myeloid-mediated CART inhibition, many studies have aimed to engineer CART cells to target and eliminate immunosuppressive macrophages in TME for better immune responses5. However, the mechanisms behind interactions between CART cells and immunosuppressive macrophages are still not fully elucidated. Additionally, there are limited publicly available xenograft models of these interactions. Therefore, there is an urgent need for models studying interactions between human macrophages and CART cells both in vitro and in mouse models.

Here, we describe a method where human CD19-targeted CART (CART19) cells are cocultured with CD19+ tumor cells and ex vivo polarized M2-like macrophages. Additionally, we report a xenograft mouse model in which immunodeficient mice are subcutaneously engrafted with both target cells and human macrophages, allowing for the evaluation of immunotherapies of interest in a setting with immunosuppressive macrophages. Specifically, we analyzed CART antigen-specific proliferation in the presence or absence of immunosuppressive macrophages in vitro, showing significant inhibition in T cell expansion in both transwell and direct co-culture plates. In our xenograft model, NSG mice were subcutaneously engrafted with tumor cells and human macrophages suspended in Matrigel for therapy screening. This animal model successfully showed that ex vivo differentiated immunosuppressive human macrophages were able to promote tumor progression in NSG mice. By following this protocol, the efficacy of immunosuppressive macrophage-targeted therapeutics can be tested, including that of small-molecule drugs, biologicals, and other cell-based therapies. However, due to limitations of using immunodeficient mice to study the human TME, the proposed models are best suited for proof-of-concept studies.

Protocol

The protocol below follows the guidelines of Mayo Clinic's Institutional Review Board (IRB), Institutional Biosafety Committee (IBC), and Department of Comparative Medicine under Institutional Animal Care and Use Committee protocol A00001767.

1. Preparation of human classical monocytes for in vitro study

  1. Isolate peripheral blood mononuclear cells (PBMCs) from healthy blood donors as previously described9. Briefly, dilute whole blood with 2% FBS in PBS at a volume ratio of 1:1, followed by PBMC isolation in density gradient medium.
  2. Isolate human classical monocytes (CD14+CD16-) from harvested PBMCs by negative magnetic bead selection according to the manufacturer's instructions (Table of Materials). Briefly, wash the appropriate number of PBMCs with isolation buffer and resuspend cells in the indicated volume according to the manufacturer's instructions. Label cells except classical monocytes with an antibody cocktail and magnetic microbeads. Collect classical monocytes from the flow-through, while retaining non-monocytes in the columns.
    NOTE: Assume 5% of the whole PBMCs are classical monocytes and adjust the starting cell number of PBMCs accordingly. Save the remaining PBMCs for purity confirmation of the monocyte population in step 1.7. Classical monocytes should always be isolated from fresh PBMCs without prior cryopreservation.
  3. Count the number of isolated monocytes using a cell counter (Table of Materials) and spin down cells at 300 x g at 4 °C for 5 min. Resuspend cells in cell isolation buffer to reach 1 x 106 cells/mL as the final concentration. Take 100 µL of cell suspension and transfer to a flat-bottom 96-well plate.
  4. Transfer 1 x 105 PBMCs into the same plate as a negative gating control to confirm classical monocyte population purity by flow cytometry. Spin down cells at 300 x g at 4 °C for 3 min. Keep isolated monocytes on ice during steps 1.5-1.12.
  5. Wash by resuspending cells with 200 µL of FACS buffer and spin down cells at 300 x g at 4 °C for 3 min. Carefully decant supernatants.
  6. For each sample, prepare 50 µL of FACS buffer containing 0.3 µL of live/dead reagent (excitation at 405 nm), 0.5 µL of APC-conjugated anti-human CD1410 (1 ng/µL), and 0.5 µL of PE-conjugated anti-human CD1611 (1 ng/µL; Table of Materials).
    NOTE: To keep staining consistent, make a master mix for multiple samples according to the total sample number. Transfer 50 µL of staining solution into each well.
  7. Add 50 µL of staining solution to each well and gently mix cells to avoid bubbles. Incubate cells in the dark at room temperature for 15 min.
  8. Add 200 µL of FACS buffer to each well and spin down cells at 300 x g at 4 °C for 3 min. Carefully decant supernatants.
  9. Wash by adding 200 µL of FACS buffer to each well and spin down cells at 300 x g at 4 °C for 3 min. Carefully decant supernatants and resuspend cells in 200 µL of FACS buffer. Run samples on a flow cytometer (Tables of Materials).
  10. Gate CD14+CD16- population. Make sure the CD14+CD16- population is above 90% of live cells for an ideal purity. Gate CD14+CD16- population of live singlets.
    NOTE: It is assumed that users are familiar with flow cytometry analysis.

2. M0 macrophage differentiation from human classical monocytes

NOTE: M2-like macrophage differentiation from human classical monocytes is adapted from a previous publication12.

  1. Spin down isolated monocytes at 300 x g at 4 °C for 5 min and aspirate supernatants. Resuspend cells in cell culture media to reach a final concentration of 1 x 106 cells/mL.
  2. Add human recombinant GM-CSF to reach a final concentration of 10 ng/mL (Table of Materials) and mix well. Transfer 100 µL of cells into a tissue culture-treated 48-well plate and add an additional 50 µL of cell culture media. Make sure that each condition has 3 replicates for the final readout on day 11.
    NOTE: Users can add more experiment groups accordingly. This protocol only includes experimental cocultures of tumor cells and T cells with or without M2-like macrophages (Table 1).
  3. Transfer 100 µL of cells into another 48-well plate and add an additional 50 µL of cell culture media for macrophage phenotyping. Make sure to include one well for phenotyping of M0 macrophages and one for phenotyping of M2-like macrophages.
  4. Add 200 µL of PBS in the surrounding wells to prevent cell culture evaporation. Incubate monocytes at 37 °C for 7 days to differentiate cells into M0 macrophages.
  5. To study interactions between differentiated macrophages and donor-matched CART19 cells, isolate 1 x 107 T cells from the PBMCs of the same donor and generate CART19 cells following an 8-day CART production procedure as previously described9,13,14.
    NOTE: CART cells costimulated with CD28 signaling domain are generated from 5 x 106 T cells as previously described9,13,14. In parallel, untransduced (UTD) T cells are generated following the same process9,13,14. UTD T cells will be used as a negative control in the T cell proliferation to control for allogeneic proliferation.

3. M2-like macrophage differentiation from M0 macrophages through coculture with target cells

  1. On day 7, take the 48-well plate with M0 macrophages and incubate on ice for at least 30 min. Vigorously pipette cells constantly while keeping the plate on ice.
    NOTE: The length of ice incubation depends on how adherent macrophages are, which can be different due to donor variance. The first mixing is to collect loosely adherent macrophages, so a 5 min pipetting should be sufficient.
  2. Transfer the detached cells to a fresh 15 mL conical tube (Table of Materials) pre-chilled on ice. Add 200 µL of ice-cold PBS to the wells of the 48-well plate and vigorously pipette remaining cells every 10 min until most macrophages are detached from the bottom. After each pipetting, check cells under a microscope and adjust ice incubation time as needed, with longer incubation times for more adherent cells.
  3. Transfer the remaining detached M0 macrophages into the 15 mL conical tube on ice. Mix cells carefully and count cells on a cell counter.
  4. Spin down cells at 300 x g at 4 °C for 5 min and aspirate supernatants. Resuspend in ice-cold PBS to reach a final concentration of 1 x 106 cells/mL.
  5. Transfer 200 µL of cells to a flat-bottom 96-well plate (Tables of Materials). Spin down cells at 300 x g at 4 °C for 3 min. Carefully decant supernatants. As a negative gating control, add an extra well stained with markers of the parent population only.
  6. Add 100 µL of FACS buffer supplemented with 1 µL of Fc receptor blocking solution to each sample. Incubate cells for 10 min at room temperature. Spin down cells at 300 x g at 4 °C for 3 min. Carefully decant supernatants.
    NOTE: It is important to pre-incubate macrophages with Fc receptor blocking buffer prior to actual marker staining to prevent false-positive results.
  7. For the sample well, prepare 50 µL of FACS buffer containing 0.3 µL of live/dead reagent (excitation at 405 nm), 0.5 µL of APC-conjugated anti-human CD1410 (1 ng/µL), 0.5 µL of APC/Cy7-conjugated anti-human CD20615 (2 ng/µL), and 0.5 µL of PerCP-conjugated anti-human CD16316 (2 ng/µL; Table of Materials). For the negative gating control, prepare 50 µL of FACS buffer containing 0.3 µL of live/dead reagent and 0.5 µL of APC-conjugated anti-human CD14.
  8. Add 50 µL of staining solution to each well and gently mix cells to avoid bubbles. Incubate cells in the dark at room temperature for 15 min.
  9. Add 200 µL of FACS buffer to each well. Spin down cells at 300 x g at 4 °C for 3 min. Carefully decant supernatants.
  10. Wash by adding 200 µL of FACS buffer. Spin down cells at 300 x g at 4 °C for 3 min. Carefully decant supernatants and resuspend cells in 200 µL of FACS buffer.
  11. Run samples on a flow cytometer and gate the CD14+CD206+CD163+ cell population. Use the expression of CD163 and CD206 on M0 macrophages as a baseline for M2-like phenotypes. Population migration from CD163lowCD206low to CD163high or CD206high is expected in M2-like macrophages. The magnitude of the population shift can be donor-dependent.
  12. Prepare the tumor cell line of interest and count on a cell counter.
    NOTE: This protocol uses the non-adherent CD19+ mantle cell lymphoma cell line, JeKo-1, as a target. Other tumor cell lines and their ability to differentiate M0 to M2-like macrophages can be tested as needed.
    1. Maintain JeKo-1 cells with the vendor's suggested culture media (Table of Materials) at 1 x 106 cells/mL in a tissue culture flask. Mix the tumor cells in the flask and count them with a cell counter.
  13. Spin down target cells at 300 x g at 4 °C for 3 min and aspirate supernatants. Resuspend target cells with cell culture media to reach a final concentration of 2 x 106 cells/mL.
    NOTE: Prepare the appropriate number of tumor cells based on the number of experimental groups. Make sure there are 2 x 105 target cells for each M2-like macrophage coculture group, including the M2 phenotyping plate.
  14. Transfer 100 µL of tumor cells to M2-like macrophage coculture groups in the 48-well plate prepared on day 0 and pipette gently to mix. Incubate cells at 37 °C for another 24 h.
    NOTE: A 2:1 ratio of JeKo-1: M0 macrophages has been tested for M2-like phenotype differentiation. Other ratios can be tested as needed.
  15. On day 8, place M2-like phenotyping plate on ice, vigorously detach cells, and prepare for phenotyping as described in steps 3.4-3.11.
    NOTE: M2-like macrophages are much more adherent than M0 macrophages, so the need for longer ice incubation is expected. This protocol only uses ice and ice-cold PBS to detach macrophages to minimize stress on the cells.
  16. Combine the flow cytometry files of the M0 phenotyping from day 7 with M2-like phenotyping. Gate CD206 and CD163 of the CD14+ parent cell population of the M0 macrophage sample and apply the same gating strategy to the M2-like macrophage sample to visualize the population shift.
  17. To introduce T cells into the cocultures, harvest day 8 CART19 and UTD T cells generated in step 2.5 and transfer the cells to 50 mL conical tubes. Mix well with a pipette and count with a cell counter.
  18. Spin down cells at 300 x g at 4 °C for 5 min and aspirate supernatants carefully. Resuspend CART or UTD T cells in cell culture media to reach a final concentration of 2 x 106 cells/mL.
  19. Prepare the tumor cell line and count on the cell counter. Spin down the cells at 300 x g at 4 °C for 5 min and aspirate the supernatants.
  20. Resuspend tumor cells with cell culture media to reach final concentration of 2 x 106 cells/mL. Transfer 100 µL of tumor cells into coculture wells without M2-like macrophages (Table 1).
  21. Transfer 100 µL of CART or UTD T cells into the proper coculture wells (Table 1). The final coculture with M2-like macrophages should have tumor cells, T cells, and macrophages at a ratio of 2:2:1. Use cocultures without macrophages as controls.
  22. To keep the final volume consistent across groups, add another 100 µL of cell culture media to wells without macrophages. Incubate cells at 37 °C for 3 days.
    NOTE: If allogeneic CART cells are being used, users must include donor-matched UTD T cells in the cocultures as a proper control to detect any allogeneity-induced T cell expansion. In this case, cryopreserved previously generated CART and UTD T cells should be thawed and recovered properly, followed by at least 3-4 h of rest prior to adding to cocultures.

4. Analysis of antigen-specific proliferation of cocultures

  1. On day 11, place the coculture plate on ice and vigorously pipette cells. Mix cells well and transfer cells into 1.5 mL microcentrifuge tubes.
    NOTE: The primary readout of the cocultures is to quantify T cell expansion, so detaching macrophages is not necessary unless users plan to analyze other markers on macrophages in the same panel as well.
  2. Add 200 µL of ice-cold PBS into the sample wells and pipette vigorously. Transfer the remaining solution to the same microcentrifuge tubes.
  3. Check wells under the microscope (10x) to make sure that suspension cells are being harvested completely. Spin down cells at 300 x g at 4 °C for 5 min and aspirate supernatants.
  4. Resuspend cells from all conditions with 200 µL of FACS buffer and transfer to a flat-bottom 96-well plate. Spin down cells at 300 x g at 4 °C for 3 min. Carefully decant supernatants.
  5. Prepare 50 µL of FACS buffer containing 0.3 µL of live/dead reagent (excitation at 405 nm) and 0.5 µL of APC-conjugated anti-human CD317 (1 ng/µL; Table of Materials) for each sample well. Add 50 µL of staining solution into each well and carefully mix the cells with a pipette to avoid bubbles.
    NOTE: In this step, there will be large cell pellets. To make sure all T cells are being stained, check the plate bottom to confirm there are no remaining cell pellets after mixing thoroughly.
  6. Incubate cells in the dark at room temperature for 15 min. Add 200 µL of FACS buffer to each well. Spin down cells at 300 x g at 4 °C for 3 min. Carefully decant supernatants.
  7. Wash by adding 200 µL of FACS buffer. Spin down cells at 300 x g at 4 °C for 3 min. Carefully decant supernatants and resuspend cells in 200 µL of FACS buffer.
  8. Run samples on a flow cytometer. Gate CD3+ cells and quantify the absolute number of live CD3+ cells as below:
    CD3 T cell count formula; equation for calculating absolute number in flow cytometry analysis.
  9. Graph the absolute number of CD3+ cells in each condition using GraphPad Prism.
    NOTE: Cocultures with M2-like macrophages are expected to have much lower absolute number of CD3 T cells than conditions in absence of M2-like macrophages.

5. Impact of M2-like macrophages on CART cells in a transwell system

NOTE: Transwell plates physically separate M2-like macrophages from stimulated CART cells and only allow soluble molecules to traffic between the cell types, letting users study dynamics of contact-independent interactions between macrophages and CART cells (Figure 1).

  1. Isolate 1 x 106 human classical monocytes and confirm population purity by flow cytometry as described in steps 1.1-1.10. Spin down 1 x 106 monocytes at 300 x g at 4 °C for 5 min and aspirate supernatants.
    NOTE: Users might want to generate autologous CART and donor-matched UTD cells as described in step 2.5.
  2. Resuspend cells in cell culture media to reach a final concentration of 1 x 106 cells/mL. Add human recombinant GM-CSF to reach a final concentration of 10 ng/mL and mix well.
  3. Transfer 200 µL of resuspended cells into the bottom well of a 24-well transwell plate (0.4 µm) with 3 technical replicates. Add an additional 100 µL of cell culture media to each well and make sure the well bottom is fully covered. Add 300 µL of cell culture media to another 3 wells for conditions without macrophages (positive control).
  4. Add 200 µL of PBS in the surrounding wells to prevent cell culture evaporation. Incubate cells at 37 °C for 7 days.
  5. On day 7, prepare 1.4 x 106 JeKo-1 tumor cells and transfer them to a 15 mL conical tube. Spin down the cells at 300 x g at 4 °C for 5 min and aspirate the supernatants.
  6. Resuspend tumor cells with cell culture media to reach a final concentration of 2 x 106 cells/mL. Add 200 µL of tumor cells into the bottom wells of all sample conditions and mix well. Incubate cells at 37 °C for another 24 h. Use bottom wells with only JeKo-1 as positive control.
  7. On day 8, harvest day 8 CART and UTD T cells generated in step 5.1. and resuspend T cells in cell culture media to reach a final concentration of 1 x 106 cells/mL.
    NOTE: If allogeneic CART cells are being used, users must include donor-matched UTD T cells in the cocultures as described in step 3.22.
  8. Count and prepare 1.4 x 106 JeKo-1 cells. Transfer tumor cells to a 15 mL centrifuge tube and spin down cells at 300 x g at 4 °C for 5 min.
  9. Aspirate supernatants and resuspend cells in cell culture media to reach a final concentration of 1 x 106 cells/mL. Transfer 100 µL of prepared T cells and 100 µL of tumor cells into each upper well for all sample conditions.
  10. Mix cells thoroughly in the upper well and incubate at 37 °C for 3 days.
  11. On day 11, mix cells in the upper chamber and transfer to 1.5 mL microcentrifuge tubes. Wash upper wells with 200 µL of PBS and transfer remaining cells into the microcentrifuge tubes.
  12. Spin down cells at 300 x g at 4 °C for 5 min and aspirate supernatants. Conduct T cell proliferation assay as described in steps 4.4-4.9.
    NOTE: JeKo-1-polarized M2-like macrophages can suppress antigen-specific T cell proliferation in a contact-independent manner. Other tumor models can be tested as needed.

6. Establishment of xenograft model with both human macrophages and tumor in NSG mice

  1. Isolate 1 x 107 human classical monocytes and confirm purity by flow cytometry as described in steps 1.1-1.10. Use NSG mice engrafted with JeKo-1 alone as a control to confirm pro-tumoral effects of M2-like macrophages, as illustrated in Figure 2.
    NOTE: This protocol engrafts NSG mice with 5 x 105 human macrophages subcutaneously. Macrophage recovery after detaching on day 7 is approximately 50%. Users can adjust cell numbers accordingly and make sure to prepare extra cells.
  2. Spin down isolated monocytes at 300 x g at 4 °C for 5 min and aspirate supernatants. Resuspend cells in cell culture media to reach a final concentration of 1 x 106 cells/mL.
  3. Add human recombinant GM-CSF to reach a final concentration of 10 ng/mL and mix well. Transfer monocytes into a T25 tissue culture flask and incubate the cells at 37 °C for 7 days.
  4. On day 7, vigorously pipette cells on ice every 10 min and check cells under a microscope until most macrophages are detached. Transfer cells into a 15 mL conical tube on ice, wash the flask with ice-cold PBS to loosen the remaining cells, and combine the cells in the conical tube.
  5. Count cells on the cell counter. Spin down cells at 300 x g at 4 °C for 5 min and aspirate supernatants.
  6. Wash and resuspend cells with 5 mL of ice-cold PBS. Spin down cells at 300 x g at 4 °C for 5 min and aspirate supernatants.
  7. Resuspend cells in ice-cold PBS to reach a final concentration of 2 x 107 cells/mL. Transfer the cell solution into a 1.5 mL microcentrifuge tube and keep it on ice.
  8. Prepare 1.5 x 107 luciferase+ JeKo-1 cells and transfer cells into a 15 mL conical tube.
    NOTE: Due to the low number of injected cells, bioluminescence imaging (BLI) is necessary to monitor tumor burden. The number of macrophages and tumor cells for subcutaneous injections can be scaled up as needed. If tumors are visible and accessible by caliper after injection, wild-type tumor cells can be used instead.
  9. Spin down cells at 300 x g at 4°C for 5 minutes and aspirate supernatants. Resuspend cells in ice-cold PBS to reach a final concentration of 4 x 107/mL.
  10. Transfer the same volume of macrophages and tumor cells into another fresh 1.5 mL microcentrifuge tube and mix well. Add the same volume of solubilized basement membrane matrix into the mixed cell solution and mix well on ice.
    NOTE: The basement membrane matrix needs to be thawed on ice overnight to prevent solidification. At this point, every 100 µL of cell solution has 5 x 105 macrophages and 1 x 106 JeKo-1 suspended in the basement membrane matrix, which uses the same cell number ratio used in the in vitro M2-like macrophage differentiation protocol.
  11. As a control, transfer the remaining prepared JeKo-1 cells with the same volume of ice-cold PBS into a fresh 1.5 mL microcentrifuge tube, adding the same volume of the basement membrane matrix to the cell solution and mixing well on ice.
  12. Prepare 10 6-8-week-old female and male NOD-SCID-γ-/- (NSG) mice (~20 g) for inhalation anesthesia with 2.5% isoflurane. Introduce isoflurane anesthesia using a rodent inhalant anesthesia apparatus. The vaporized isoflurane is delivered into a chamber and a heated stage with nose cones.
  13. Place mice into the anesthetic chamber for 1-2 min. Transfer mice to the nose cones on the heated stage once postural and right reflexes are lost18. Keep mice on the heating stage throughout the procedure. Apply eye ointment to both eyes of the mice to prevent corneal damage due to the loss of the blink reflex during anesthesia.
  14. Carefully shave the right flank of each mouse to expose the skin after the mice have been anesthetized. Load a 0.5 mL syringe with 100 µL of JeKo-1-macrophage cell mixture and carefully remove bubbles in the solution.
  15. Carefully lift the exposed skin by hand and insert the needle into the lifted region. Keep lifting the skin with the needle and press one finger at the injection site. Try not to move the needle once inserted to prevent accidental punctures and cell leakage.
  16. Slowly release the cells into the subcutaneous layer and carefully remove the needle. Repeat the subcutaneous injections for all 10 NSG mice, with 5 mice receiving both JeKo-1 and macrophages and 5 mice receiving JeKo-1 alone.
  17. Prepare D-luciferin (Table of Materials) at a concentration of 15 mg/mL. Load a 0.5 mL syringe with 200 µL of D-luciferin and inject into each mouse intraperitoneally.
  18. Place anesthetized mice on the stage for BLI 10 min after D-luciferin injection. Perform BLI for each mouse and record as baseline (Day 0) tumor burden. Place the mice back into their cages.
    NOTE: Users need to ensure that the anesthetized mice are not left unattended until they regain consciousness to maintain sternal recumbency after the imaging procedure.
  19. Monitor tumor burden by BLI 2x per week until experimental or humane endpoints are reached. Overall survival endpoint is reached once tumor volume is at or above 2000 mm3, calculated by formula below:
    Tumor volume calculation, formula: Width*Length*Height/2; mathematical expression.
    NOTE: The length of tumor monitoring can be adjusted based on the purpose of the experiments and desired readouts. Due to extremely small tumor sizes in the first 2-3 weeks, BLI is recommended for tumor burden indication. Once tumors become visible and accessible by caliper, both BLI and caliper measurements are recommended for accurate tumor analysis.
  20. To study interactions between engrafted human macrophages and autologous CART19 cells in NSG mice, generate and engraft ex vivo-differentiated human macrophages as described in steps 6.1-6.16. Isolate autologous T cells from the same blood donor in step 6.1 following the 8-day CART manufacturing protocol as previously described9,13,14.
    NOTE: Each NSG mouse is treated with 2 x 106 CART19 cells. Users should generate at least 2 x 107 CART cells to treat 10 mice. CART cells are cryopreserved on day 8 and stored until JeKo-1/macrophages are engrafted in the mice, 5 days after subcutaneous injections.
  21. After 5 days of tumor/macrophage injections, randomize mice based on BLI as described in steps 6.17-6.18. Thaw and prepare CART cells as described in step 3.22. Wash CART cells with PBS and spin down cells at 300 x g at 4 °C for 5 min.
  22. Resuspend CART cells in PBS to reach a final concentration of 2 x 107 cells/mL. Load a 0.5 mL syringe with 100 µL of prepared CART cells and inject into each tumor-engrafted mouse intravenously through the tail vein.
  23. Monitor tumor burden by BLI once a week for 4 weeks.
    NOTE: Due to the immunosuppressive functions of engrafted human macrophages, impaired CART antitumor activities and higher tumor burden are expected in mice injected with JeKo-1/macrophages.

7. M2-like phenotype confirmation in the engrafted human macrophages by immunofluorescence staining

  1. To confirm JeKo-1-induced M2-like phenotype in engrafted human macrophages described in step 6 by immunofluorescence staining, follow steps 6.1-6.19 and scale up the number of injected macrophages and luciferase+ JeKo-1 to 5 x 106 cells and 1 x 107 cells per NSG mouse, respectively.
    NOTE: Users should make sure that the final volume of injection for each mouse is 100 µL.
  2. On day 7, euthanize NSG mice by CO2 (​set the flow meter to 3.5 L/min for 5 min or until animals are euthanized), perform cervical dislocation, and confirm loss of corneal reflexes as previously described19. Place mice on the work bench with tumors facing upwards. Spray 70% ethanol on the tumor sites and carefully harvest engrafted tumors with scissors and forceps.
  3. Transfer tumor tissues into 50 mL conical tubes filled with 20-30 mL of 4% paraformaldehyde for fixation as previously described20. Cryopreserve and cryosection tumor tissues for immunofluorescence staining according to a previously published protocol20.
  4. Stain tumor tissue slides with mouse-anti-human CD206 antibody conjugated with AF546 (1:50) and rabbit-anti-human iNOS antibody conjugated with AF647 (1:100) diluted in immunofluorescence staining buffer, followed by nucleus staining and preservation as previously described21.
    NOTE: For optimal staining results, users should perform antibody titration accordingly, especially when different tumor cell lines are being used.
  5. Acquire immunofluorescence images with confocal microscopy at 40x. To confirm that engrafted human macrophages develop an M2-like phenotype, expect to see expression of human CD206 and minimal expression of human iNOS.

Results

The aim of this protocol is to establish both in vitro and in vivo models to study interactions between human immunosuppressive macrophages and CART cells due to the complex nature and dynamics of the interactions and the limited existing xenograft models. Following the protocol, isolated human classical monocytes are differentiated to acquire M2-like phenotypes and become functionally immunosuppressive.

To confirm the purity of isolated human classical monocytes, run stained samples on a flow cytometer and analyze by gating on the human CD14+CD16- population (Figure 3A-B). Successful monocyte isolation will give at least 90% CD14+CD16- population from the live cell parent population. To phenotype and confirm differentiated M2-like macrophages, a population shift from CD163low CD206low to CD163high or CD206high is expected when comparing M0 macrophages with M2-like macrophages (Figure 4). A successful demonstration of CART antigen-specific proliferation negatively affected by immunosuppressive macrophages should show a significant reduction in CART proliferation (Figure 5A). According to our data, JeKo-1-differentiated macrophages are also capable of inhibiting CART antigen-specific proliferation in a contact-independent manner in a transwell assay (Figure 5B). A successful xenograft model in NSG mice engrafted with human immunosuppressive macrophages should show much faster tumor progression than mice engrafted with tumor cells alone (Figure 6). To further confirm M2-like phenotype in engrafted human macrophages in NSG mice, expression of human CD206 and minimal expression of human iNOS should be observed in the macrophages by immunofluorescence staining (Figure 7A-D). Engrafted immunosuppressive human macrophages are expected to impair the CART19 in vivo antitumor activities in the NSG mice (Figure 8).

CART19, JeKo-1 interaction diagram; macrophage experiment; cell culture; immunotherapy research.
Figure 1: Schema of transwell assay studying contact-independent interactions between CART cells and macrophages. Classical monocytes are differentiated into M2-like macrophages in the bottom chamber of a transwell plate as described in the protocol. CART19 cells and JeKo-1 are cocultured at a ratio of 1:1 in the upper well for 3 days, followed by CART antigen-specific proliferation analysis by flow cytometry. Please click here to view a larger version of this figure.

Mouse tumor model diagram comparing immunosuppressive macrophages and tumor alone, subcutaneous.
Figure 2: Schema of xenograft NSG mouse model engrafted with human immunosuppressive macrophages and tumor cells. Classical monocytes are differentiated into M0 macrophages as described in the protocol. M0 macrophages are carefully detached and mixed with luciferase+ JeKo-1 at a ratio of 1:2 in Matrigel. As a control group, luciferase+ JeKo-1 are mixed with PBS at a volume ratio of 1:1 in Matrigel. NSG mice are subcutaneously engrafted with both tumor cells and macrophages or with tumor cells alone. Please click here to view a larger version of this figure.

Flow cytometry diagram analyzing live cell populations via FSC-SSC, huCD16, huCD14 markers.
Figure 3: Classical monocyte population purity confirmation by flow cytometry. Human classical monocytes (CD14+CD16-) are isolated from fresh PBMCs by magnetic negative bead selection following the manufacturer's instructions. Both PBMCs and isolated monocytes are processed for flow cytometry staining as described in the protocol. (A) Gating strategy of stained human PBMCs as a control. (B) Gating strategy of stained purified human classical monocytes. Representative flow results and gating strategies are shown. Ideal classical monocyte isolation should have >90% purity. Please click here to view a larger version of this figure.

Flow cytometry histogram of CD163, CD206 markers on macrophages; immunophenotyping comparison.
Figure 4: Analysis of M2-like phenotypes in macrophages after differentiation. M0 macrophages and JeKo-1-differentiated M2-like macrophages are prepared for CD163 and CD206 expression by flow cytometry as described in the protocol. A representative population shift is shown as a histogram half-overlay. Please click here to view a larger version of this figure.

Bar graph showing live CD3 cell count in JeKo-1+UTD vs JeKo-1+CART19, with significance markers.
Figure 5: M2-like macrophages inhibit CART19 antigen-specific proliferation in a contact-independent manner. (A) JeKo-1, M2-like macrophages, and T cells are cocultured at a ratio of 2:1:2 for 3 days as described in the protocol. The absolute number of live CD3+ cells is measured by flow cytometry. A representative proliferation analysis is shown. Mean and SEM are shown. A total of 3 biological replicates were analyzed. Two-way ANOVA is used, *p < 0.05, ***p < 0.001. (B) M2-like macrophages are physically separated from antigen-stimulated CART19 cells in a transwell plate (0.4 µm) as described in the protocol. A representative proliferation analysis is shown. Mean and SEM are shown. A total of 3 biological replicates were analyzed. Two-way ANOVA is used; **p < 0.01, ****p < 0.0001. Please click here to view a larger version of this figure.

Tumor growth chart showing JeKo-1 with macrophages vs. JeKo-1 over 17 days, displaying significance.
Figure 6: Differentiated macrophages promote subcutaneous tumor progression in NSG xenograft. NSG mice are subcutaneously engrafted with classical monocyte-differentiated M0 macrophages combined with JeKo-1 or JeKo-1 cells alone as described in the protocol. Tumor burden over time is monitored by bioluminescence imaging (BLI). Mean and SEM are shown. Two-way ANOVA is used, ***p < 0.001. Mϕ = macrophage. Please click here to view a larger version of this figure.

Immunofluorescence microscopy showing huCD206, iNOS, GFP, and merged images in cell samples.
Figure 7: Confirmation of M2-like phenotypes in engrafted human macrophages in NSG mice. NSG mice are subcutaneously engrafted with 5 x 106 ex vivo differentiated human macrophages and 1 x 107 luciferase+ JeKo-1. Tumors are harvested and prepared for immunofluorescence staining 7 days after tumor/macrophage injection. (A) Human CD206 or (B) iNOS is stained with mouse-anti-human antibody conjugated with AF547 (red) or rabbit-anti-human antibody conjugated with AF647 (magenta), respectively; (C) luciferase+ JeKo-1 generated in the lab expresses GFP as well (green); (D) DAPI is presented in blue. Please click here to view a larger version of this figure.

Tumor burden progression graph; CART19 treatment effectiveness, statistical significance marked.
Figure 8: Immunosuppressive macrophage-mediated inhibition of CART19 in vivo antitumor activities. NSG mice are injected subcutaneously with 5 x 105 human ex vivo polarized macrophages and 1 x 106 luciferase+ JeKo-1 or 1 x 106 luciferase+ JeKo-1 alone. Mice are treated with 2 x 106 CART19 intravenously by tail vein 5 days after tumor injections. Tumor burden is indicated by BLI for 4 weeks. Mean and SEM are shown. Two-way ANOVA is used, *p < 0.05. Mϕ = macrophage. Please click here to view a larger version of this figure.

48-well plate assay Transwell assay
JeKo-1+M2+CART19JeKo-1+M2+CART19
JeKo-1+CART19JeKo-1+CART19
JeKo-1+M2+UTDJeKo-1+M2+UTD
JeKo-1+UTDJeKo-1+UTD

Table 1: CART19, macrophage, and JeKo-1 coculture conditions of interest. In the 48-well coculture assay, CART19 or UTD cells were cocultured with M2-like macrophages and JeKo-1 cells at a ratio of 2:1:2 for 3 days. In the transwell assay, M2-like macrophages were seeded in the bottom chamber while JeKo-1-stimulated CART19 or UTD cells were seeded in the upper chamber.

Discussion

CART cell therapy has shown numerous clinical successes in treating subsets of patients with hematological malignancies, but most patients relapse within 1-2 years after CART infusion1,2. Various potential CART resistance mechanisms have been identified, and immunosuppressive myeloid cells are found to play an important role in TME immunosuppression and subsequent CART failure3,4,5,6,7,8. Studies have engineered CART cells to target immunosuppressive macrophages and improve overall antitumor efficacy5. However, the mechanisms behind macrophage-derived inhibition of CART are not fully understood. In addition, publicly available xenograft models studying the impacts of human macrophages on CART cells are limited due to the complexity of engrafting human macrophages in mouse models22,23. Immunodeficient mice receiving human CD34+ hematopoietic stem cells (HSCs) after irradiation showed the capability of human monocytes/macrophages engraftment22. Human CD34+ HSC-engrafted NSG-SGM3 mice indicated elevation of human myeloid cells, which have been used to study the adverse effects of CART cell therapies23. However, the process of obtaining and enriching human HSCs can be time-consuming and expensive, and the engraftment efficiency can be limited23. Here, we established more simplified in vitro and in vivo models to study the impacts of immunosuppressive macrophages on CART cells, allowing users to test cancer immunotherapies and potentially macrophage-targeted therapies in a more cost-effective manner. This model has been used to determine the effects of M2-like macrophage-derived IL-1 receptor antagonist (IL-1ra) on CART19 antitumor activity in MCL24. In this context, immunodeficient mice engrafted with MCL cells in combination with human macrophages received treatment of CART19 combined with IL-1ra neutralizing antibody or control IgG and were followed for antitumor activity for 56 days24.

The techniques in this protocol are generally straightforward, facilitating reproducibility. However, there are certain factors that are essential for a successful readout. Proper detachment of macrophages is important to obtain cells with high viability. Keeping harvested macrophages on ice throughout the process is crucial to maintain cell viability. Regarding macrophage phenotyping, collecting as many macrophages as possible from the plate is important to have a more accurate picture of the macrophage phenotype, given that not all the cells are being differentiated into the exact same state. Because macrophages express high levels of Fc receptors, incubating macrophages with Fc receptor blocking buffer before staining for biomarkers can avoid non-specific staining and false positive results on flow cytometry.

This protocol has been tested multiple times in mantle cell lymphoma models. If other tumor models are of interest, users should first confirm if the tumor cell line of interest can polarize macrophages to immunosuppressive phenotypes before performing functional assays. Additionally, the number of cells seeded in cocultures or engrafted in NSG mice can be scaled up accordingly. The limitation of this protocol is that our in vivo study relies on immunodeficient mice, which do not reflect the dynamics between additional immune cells but is still well-suited for proof-of-concept studies. This protocol allows for the study of human CART cells and human macrophages, which is of translational relevance.

Overall, this established model can be used to evaluate the efficacy of different cancer immunotherapies or therapies targeting macrophages, such as small-molecule drug screening, cell-based therapies, and other therapeutic platforms.

Disclosures

SSK is an inventor on patents in the field of CAR immunotherapy that are licensed to Novartis (through an agreement between Mayo Clinic, University of Pennsylvania, and Novartis), MustangBio (through Mayo Clinic), and Sendero (through Mayo Clinic). RLS and SSK are inventors on patents in the field of CAR immunotherapy that are licensed to Humanigen (through Mayo Clinic). KY, RLS, TH, BK, ES, and SSK are inventors on patents in the field of CAR immunotherapy that are licensed to Immix. SSK receives research funding from Kite, Gilead, Juno, BMS, Novartis, Humanigen, MorphoSys, Tolero, Sunesis/Viracta, LifEngine Animal Health Laboratories Inc., Incyte, and Lentigen. SSK has participated in advisory meetings with Kite/Gilead, Humanigen, Juno/BMS, Capstan Bio, and Novartis. SSK has served on the data safety and monitoring board with Humanigen and Carisma. SSK has served as a consultant for Torque, Calibr, Novartis, Capstan Bio, Carisma, and Humanigen.

Acknowledgements

This study was partly funded through the Mayo Clinic Comprehensive Cancer Center (SSK), the Mayo Clinic Center for Individualized Medicine (SSK), the Mayo Clinic Center for Regenerative Biotherapeutics (SSK), the National Institutes of Health grant R37CA266344-01 (SSK), and the Department of Defense grant CA201127 (SSK).

Materials

List of materials used in this article
NameCompanyCatalog NumberComments
0.5 mL syringeFisher Scientific329461
1.5 mL microcentrifuge tubeEppendorf1415-2508 
15 mL conical tubeCorning Life Sciences352097
24-well transwell plate (0.4 μm)Corning Life Sciences3397
50 mL conical tubeCorning Life Sciences352098
APC/Cy7-conjugated anti-human CD206Biolegend321119Flow antibody
APC-conjugated anti-human CD14Biolegend325607Flow antibody
APC-conjugated anti-human CD3Biolegend344811Flow antibody
autoMACS Rinsing SolutionMiltenyi Biotec130-091-222Classcial monocyte isolation buffer 
Basement Membrane MatrixCorning Life SciencesCLS356237Solubilized basement membrane matrix
Cell culture media10% human AB serum 1% penicillin-streptomycin-glutamine in X-VIVO 15 Serum-free Hematopoietic Cell Medium
Classical monocyte isolation kit, humanMiltenyi Biotec130-117-337
CytoFLEX System  B5-R3-V5Beckman CoulterC04652Flow cytometer
D-luciferinGold BiotechnologyLUCK-1G
D-PBS (Dulbecco's phosphate-buffered saline)Gibco14190250 
Eye ointmentThermoFisher19-090-832
FACS buffer2% FBS 1% sodium azide in PBS
Fetal bovine serumCorning Life Sciences35-011-CV
Flat-bottom 96-well plateCorning Life SciencesCLS3997
Heat inactivated pooled human AB serum Innovative Research4931
Human TruStain FcX (Fc Receptor Blocking Solution)Biolegend422301
Isoflurane liquid  Piramal Pharma Limited66794-017-10
IVIS Spectrum In Vivo Imaging SystemPerkinElmer124262
JeKo-1 cell line culture media10% FBS 1% penicillin-streptomycin-glutamine in RPMI-1640 medium
Live/dead Fixable Aqua Dead Cell Stain Kit, for 405 nm excitationInvitrogenL34966
LS columnMiltenyi Biotec130-042-401Negative bead selection
LymphoprepStemcell Technologies07811Density gradient medium
MACS BSA Stock SolutionMiltenyi Biotec130-091-376Classcial monocyte isolation buffer 
Mouse-anti-human CD206 antibody conjugated with AF546 Santa Cruz Biotechnologysc-376232
NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) miceJackson Laboratory 5557
PE-conjugated anti-human CD16Biolegend302007Flow antibody
Penicillin-Streptomycin-Glutamine (100x), LiquidGibco10378-016
PerCP-conjugated anti-human CD163Biolegend333625Flow antibody
Rabbit-anti-human iNOS antibody conjugated with AF647 ProteintechCL647-18985
Recombinant human GM-CSFStemcell Technologies78015
RPMI-1640 mediumGibco11875093
SepMate-50 IVDStemcell Technologies85450PBMC isolation by density gradient centrifigation 
Sodium Azide, 5% (w/v)Ricca Chemical7144.8-16
Tissue-culture treated 48-well plateCorning Life Sciences3548
Vi-cell XR Cell Viability AnalyzerBeckman CoulterCell counter
X-VIVO 15 Serum-free Hematopoietic Cell MediumLonza04-418Q

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Myeloid Cell InteractionsImmunosuppressive MacrophagesTumor MicroenvironmentIn Vitro CocultureIn Vivo XenograftHematopoietic Stem CellsMacrophage EngraftmentBioluminescence ImagingMacrophage Targeted Immunotherapy

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