A Simple and Efficient Method for Testing Immunomodulatory Agents for Generation of Tolerogenic Dendritic Cells from Human CD14⁺ Monocytes

We seek to understand what immunomodulatory treatments can generate tolerized dendritic cells from already differentiated monocyte-derived dendritic cells, and this is very valuable for autoimmune and transplantation research. Self therapies are becoming more practical due to advancements in manufacturing and tolerogenic dendritic cells have shown promise in preclinical studies. They can generate antigen specific tolerance. The most common is for cytometry. This provides a rapid and also accessible way to analyze tolerized cells. It is challenging to generate tolerogenic dendritic cells that persist in both function in vivo. This is why there are no clinically approved dendritic cell therapies for tolerized. We have identified novel combinations of immunomodulatory compounds from large screening studies that these show improvement in tolerized dendritic cell functionality. We also engineer tolerizing nanoparticle formulations.

[Instructor] To begin, place the 15 milliliter tubes containing enriched human monocytes and with T-cells in a centrifuge. After centrifugation is complete, use a pipette to aspirate the supernatant from the tubes. Add one milliliter of MODC culture medium to the monocyte pellet, one milliliter of T-cell culture medium to the tube with the T cells and mix well before cell counting. Next, add nine milliliters of warm MODC culture medium to the monocyte suspension to make the final volume of 10 milliliters. Then pipette 100 microliters each of GM-CSF and IL-4 stock solutions. Transfer the suspension to a labeled Petri dish, marked as MODC, and incubate. Next, add one milliliter of T-cell freeze medium to the T-cell suspension. Divide the mixture into two two milliliter cryo vials. Place the sealed cryo vials in a freezing chamber with balancing tubes in unused wells. On day four, add five milliliters of fresh MODC culture medium to the monocyte tube. Then pipette 100 microliters each of GM-CSF and IL-4 stock solutions seven and incubate until day seven. On day seven, transfer the differentiated monocyte-derived dendritic cells, or MDOCs, into a 50 milliliter tube and centrifuge as before. Add one milliliter of warm MODC culture medium to the cell pellet and pipette up and down to mix well. Count the cells using a hemocytometer. Dilute the suspension with warm MODC culture medium containing GM-CSF and IL4 until the desired cell concentration is obtained. Dispense 100 microliters of the suspension containing 30,000 cells into each well of a flat-bottom 96 well tissue culture plate. Add one microliter of immunomodulatory drugs into the designated wells and incubate. The next day, centrifuge the MODC plate at 300 G for five minutes. After gently removing the supernatant, gently add 200 microliters of warm HBSS to the side of the plate and centrifuge. Again, add 100 microliters of warm MODC culture medium containing GM-CSF and IL4 after supernatant aspiration. If immunostimulation is used, add one microliter of 100X stock of lipopolysaccharide to the designated wells and incubate. On day eight, thaw two cryo vials in a hot bead bath at 37 degrees Celsius until the contents begin to melt. Once partially thawed transfer the vials to a cell culture hood. Then add one milliliter of warm T-cell culture medium to rapidly thaw the cells. Transfer the contents into a 50 milliliter tube and rinse the cryo vials with an additional one milliliter of T-cell culture medium to collect all cells. Top up the suspension with flow staining solution to 15 milliliters and centrifuge. Resuspend the pellet in five milliliters of flow staining solution and centrifuge again, then resuspend the cell pellet in one milliliter of warm T-cell culture medium after pipetting out the supernatant. Add nine milliliters of warm T-cell culture medium to make a final volume of 10 milliliters. Transfer the suspension to a Petri dish labeled as Rethawed T-cells and incubate. On day eight, centrifuge the MODC plate at 300 G for five minutes. After pipetting out the supernatant, add 200 microliters of flow staining solution to each well and incubate. Then pipette up and down to remove attached cells and the cell suspension to a new 96 well V-bottom plate before centrifuging again. Pipette 50 microliters of FC receptor binding inhibitor antibody into each well after aspirating the supernatant to block unspecific binding. After incubating the plate at room temperature for 30 minutes, add 50 microliters of the prepared validation panel antibody cocktail into each well. Mix 50 microliters of compensation beads with 50 microliters of each diluted antibody in 1.5 milliliter tubes and incubate. Centrifuge the plate at 300 G for five minutes. Resuspend the pellet in 200 microliters of flow staining solution to wash the cells. After the final wash and centrifugation, resuspend the cells in 110 microliters of flow staining solution for flow cytometric analysis. For the tolerogenic MODCs, substitute the antibody cocktail with the tolerance panel cocktail. On day nine, transfer the rethawed T-cell Petri dish into a 50 milliliter tube and top up the tube with flow staining solution. Use the second tube containing unstained T-cell for trig analysis. Spin the tubes at 250 G for five minutes. After removing the supernatant, resuspend the cells in warm cell culture media. Count the T-cell and ensure a minimum of 4 million cells are obtained. Centrifuge the MODC plate at 300 G for five minutes. After aspirating the supernatants, add 200 microliters of T-cells into each well. Add unstained T cells for trig analysis plates. Add 25 microliters per milliliter of anti-CD3/CD28 antibody cocktail to all groups except negative control wells to stimulate T-cells and incubate until day 12. Next, transfer all cell suspensions from the 96 well culture plate into a V-bottom plate with flow staining solution. Wash the cells twice in 200 microliters of flow staining solution. Set aside some cells for compensation controls. After the second wash, centrifuge the plate. Then add 50 microliters of diluted FC receptor binding inhibitor antibody into each well and incubate. When incubation is complete, add 50 microliters of the prepared trig panel antibody cocktail into each well. For compensation controls, mix 50 microliters of unstained compensation cells with 50 microliters of each single stained antibody. Incubate the plate and compensation controls at four degrees Celsius for one hour, then wash with flow staining solution before centrifuging. Now stain the cells with Live/Dead Near-IR dye diluted in HBSS at 200 microliters per well before cold incubation. Wash and centrifuge the plate before staining with Fox P3 and flow cytometric analysis. On day one, undifferentiated monocytes were predominantly HLA-DR-minus with a small CD14 minus, while differentiated monocytes on day seven showed most cells as HLA-DR-plus CD14 minus, CD1c-plus, CD141 plus. Treatment with rapamycin both with and without lipopolysaccharide, significantly reduced DC-SIGN-plus CD1c-plus populations and inhibited the upregulation of CD86 and CD40 markers observed with LPS treatment alone. FOX P3-plus T regulatory cell populations were increased when MDCs were co-culture with T-cells. But no significant differences were observed among the four MODC treatment groups. T-cell proliferation was reduced in both CD4-plus and CD8-plus T-cell populations after co-culture with Rapa-treated MDOCs. Interleukin-10 treated samples significantly increased Interleukin-10 production at 24 hours. Dex-treated MDOCs significantly increased interleukin-10 production, but only after resting for 72 hours. Pretreatment of dexamethasone reduced the average TNF alpha generation upon LPS treatment at 24 hours. Significant increases in PDL1-plus MDOCs were observed in the Dex plus LPS and Rapa treated groups at 72 hours. Suppression of CD86 expression was observed from all immunomodulator treated groups at both 24 and 72 hours. Immunomodulator treated MDOCs did not increase CD4-plus T-cell proliferation.