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

Our study provides simplified in vitro and in vivo models to understand the impact of human immunosuppressive macrophages on CA 19 anti-tumor activity in the context of mantle cell lymphoma. The most commonly used model to study the human macrophages and monocytes in vivo is to engraft hematopoietic stem cells into eradicated immunodeficient mice. Engrafting immunodeficient mice with human hematopoietic stem cells to study human myeloid cells in mice can be time-consuming and expensive.

The engraftment efficiency can be limited as well. We establish more simplified in vitro and in vivo models to study interactions between human macrophages, CAR T, and tumor cells. It can be used to test other macrophage-targeted immunotherapies as well.

To begin, centrifuge the isolated 10 million human classical monocytes at 300g for five minutes at four degrees Celsius. Aspirate the supernatant and resuspend the cells in culture media to a final concentration of 1 million cells per milliliter. Add human recombinant GM-CSF to the suspension to reach a final concentration of 10 nanograms per milliliter, and mix thoroughly.

Transfer the monocytes into a T25 tissue culture flask, and incubate them at 37 degrees Celsius for seven days. On day seven, pipette the cells vigorously on ice every 10 minutes, checking under a microscope until most macrophages detach. Transfer the detached cells into a 15-milliliter conical tube on ice.

Then, wash the cells with ice-cold PBS to loosen the remaining cells and combine them in the conical tube. Count the cells using an automated cell counter and centrifuge them at 300g for five minutes at four degrees Celsius. Aspirate the supernatant, and then wash and resuspend the pellet with five milliliters of ice-cold PBS.

After centrifuging the tube again and aspirating the supernatant, resuspend the final cell pellet in ice-cold PBS to reach a concentration of 20 million cells per milliliter. Transfer the solution to a 1.5 milliliter microcentrifuge tube and keep it on ice. Next, transfer the prepared 15 million luciferase-positive JeKo cells into a 15-milliliter conical tube.

After centrifuging the JeKo cells, aspirate the supernatant. Resuspend the pellet in ice-cold PBS to a concentration of 40 million cells per milliliter. Now, mix the same volumes of the macrophage suspension and tumor cell suspension in a fresh 1.5 milliliter microcentrifuge tube.

Add the same volume of solubilized basement membrane matrix and mix thoroughly on ice. As a control, mix the remaining JeKo cells with the same volume of PBS in a new microcentrifuge tube. Add the same volume of solubilized basement membrane matrix and mix on ice.

After anesthetizing the mice, place them on a heated stage with nose cones. Apply an ophthalmic ointment to both eyes of the mice to prevent corneal damage. Shave the right flank of each mouse to expose the skin.

Load 100 microliters of the JeKo macrophage mixture into a 0.5 milliliter syringe and remove air bubbles. Gently lift the exposed skin and insert the needle into the raised skin area, while applying finger pressure to the site. Slowly inject the cells into the subcutaneous layer and gently withdraw the needle.

To evaluate the in vivo impact of immunosuppressive macrophages, tumor burden progression was tracked by bioluminescence imaging in NSG mice, engrafted with JeKo cells alone or in combination with differentiated macrophages. The presence of differentiated macrophages markedly accelerated tumor progression in vivo as indicated by a significantly greater tumor burden in co-engrafted mice by day 17 compared to JeKo alone.