Novel Protocol for Generating Physiologic Immunogenic Dendritic Cells

Physiologic dendritic antigen-presenting cells, termed PhDC, are the immune master-switches which initiate antigen-specific T cell immunity and tolerance. This paper delineates an efficient method for the production of PhDC. Clinical use of dendritic cells is impeded by the non-physiologic cytokine methods used to produce them.

Our method overcomes that problem via efficient platelet signaling of monocyte-to-DC conversion in humans and mice. These PhDC are not dependent on cytokine conditions unavailable in vivo, so they can generate potent clinical anti-tumor T cell responses in both humans and mice with established cancers. Species-independent platelet initiation of monocyte-to-PhDC maturation has broad experimental applicability.

PhDC permit dissection of dendritic cell physiology, facilitate induction of therapeutic anti-cancer immunity to immunogenic cancers, and offer promise for personalized antimicrobial vaccination. This experimental system is novel, and multiple aspects of it such as coating of the plastic chamber, tumor cell treatment with 8-MOP/UVA, flow conditions, and release of monocytes are best visually represented. Demonstrating the procedure will be Eve Robinson, a technician from my laboratory.

To collect peripheral blood mononuclear cells, first set up a 15-milliliter tube with four milliliters of lymphocyte isolation medium per five to 10 mice bled. Then slowly layer blood collected from tumor-bearing mice on top of the medium. Spin the cells at 1, 000-to 1, 500-times-G for 20 minutes to separate the PBMCs from red blood cells.

At the end of centrifugation, collect the top plasma layer, leaving 0.5 milliliters remaining above the buffy coat, and store at four degrees Celsius for later use. Next, collect the buffy coat layer into a clean 15-milliliter tube filled with PBS to 15 milliliters, and spin at 250-times-G in a standard tissue culture centrifuge for 10 minutes to collect the PBMC. At the end of centrifugation, carefully pipette off the supernatant and flick the tube to resuspend the pellet.

Then add two milliliters of ACK red blood cell lysis buffer to the tube to remove any remaining red blood cells from the PBMC. Incubate the PBMC on ice for 10 minutes. Fill the tube with PBS to 15 milliliters, and spin down at 250-times-G in a standard tissue culture centrifuge for 10 minutes to collect the PBMC.

Carefully pipette off the supernatant and flick the tube to loosen the pellet. Resuspend the pellet in 300 microliters of FBS. To prepare YUMM1.7 tumor cells per treatment group, first resuspend the tumor cell pellet in FBS at 2.5 million cells per 300 microliters.

With tissue culture hood lights off, add 8-methoxypsoralen for a final concentration of 100 nanogram per milliliter to the YUMM1.7 tumor cell suspension. Next, mix the cells, wrap the cell container in foil, and incubate at 37 degrees Celsius for 20 minutes. To pre-coat a 12-well tissue culture plate, add one milliliter of FBS to one well for every treatment group of five mice, and refrigerate the plate at four degrees Celsius for 20 minutes.

After 20 minutes, under a tissue culture hood, remove FBS from the wells, and add 300 microliters of methoxypsoralen-exposed tumor cells per well. Then expose the cell-containing plate to the pre-warmed ultraviolet A light source for total irradiation of four joules per square centimeter. To collect 8-methoxypsoralen/UVA-treated tumor cells from each well, swirl the plate and pipette carefully to ensure complete cell recovery.

To perform transimmunization plate passage for each treatment group of five mice, combine 300 microliters of the appropriate PBMC and 300 microliters of 8-methoxypsoralen/UVA-treated tumor cells in a 1.5-milliliter conical tube and mix the cells. Next, open the entrance and exit tubing clamps of the TI plate. Use a 10-milliliter syringe to fill the TI tubing with FBS, closing the clamps as the tubing is filled to retain FBS inside the tubing.

Disconnect the syringe. Place a P1000 pipette tip securely into the TI plate intake. Hold the plate at a 45-degree angle and fill the plate slowly with PBMC and tumor cell mix, avoiding bubbles.

Remove the pipette tip from the intake before releasing the pipette plunger. Return the remaining cells to the 1.5-milliliter conical tube. Incubate the FBS-filled tubing, the cell-filled TI plate, and the 1.5-milliliter conical tube containing the remaining cells in the tissue culture incubator at 37 degrees Celsius for one hour.

After incubation, release the clamps and empty the TI tubing by gravity. Then insert a P1000 pipette tip with the plunger depressed into the plate port to remove the cells from the TI plate. Hold the plate at a 45-degree angle and fill the P1000 pipette tip.

Place cells back into their original 1.5-milliliter conical tube. To run the TI plate, connect the exit tube to the plate, and secure the TI plate into the plate-running system. Next, using a one-milliliter syringe, draw up those 600 microliters of PBMC and tumor cell mix from the 1.5-milliliter conical tube.

Attach the syringe to the entrance tube with the clamp open, and slowly fill until fluid reaches the end of the tubing. Attach the free end of the entrance tube to the TI plate and continue gently loading the remaining volume. Close the entrance clamp when finished.

Detach the one-milliliter syringe and connect the entrance tubing to the syringe pump. Secure the exit tubing to a clean 1.5-milliliter conical tube for cell collection. Adjust the syringe pump flow rate to 0.09 milliliters per minute.

Tilt the TI plate approximately 30 degrees towards the syringe pump side using a TI plate running platform. Carefully release the clamp on the entrance tubing. Start the syringe pump, observing carefully how the TI plate fills.

Once the TI plate is completely filled, tilt it approximately 30 degrees in the opposite direction as it empties. When all the cells in the liquid are in the 1.5-milliliter conical collection tube, stop the pump. To wash the TI plate, disconnect the entrance tubing from the syringe pump, and connect it to a one-milliliter syringe filled with 600 microliters of FBS.

Fill until FBS is fully loaded. Then disconnect the syringe and attach the tubing to the syringe pump. Adjust the syringe pump flow rate to 0.49 milliliters per minute.

Release the entrance clamp, and run the TI plate, collecting the wash in the same 1.5-milliliter conical tube. Flick or tap the TI plate gently the entire time to aid in detaching and eluting any adherent cells as the plate empties. Disconnect the collection 1.5-milliliter conical tube from the TI plate setup.

Spin the collection tube in the benchtop microfuge at 250-times-G for eight minutes and discard the supernatant. To perform overnight co-incubation of PBMC with antigen, first resuspend the PBMC and tumor cell pellet in two milliliters of clear RPMI supplemented with 15%autologous mouse serum. Then plate cells in a 35-millimeter non-tissue-culture treated sterile dish, and incubate overnight under standard tissue culture conditions.

On the following day, carefully detach any adherent cells from the bottom of the dish with a tissue culture scraper. Rotate the dish while scraping to ensure even cell collection. Collect the cells in a 15-milliliter tube.

Add two milliliters of PBS to the dish, and repeat the scraping step, collecting the cells into the same tube. Rinse the 35-millimeter dish with one milliliter of PBS, and add the rinse to the same tube. Spin at 250-times-G in a standard tissue culture centrifuge for 10 minutes to collect the cells.

Carefully pipette off the supernatant, then flick the tube to resuspend the pellet. The therapy showed reduction of YUMM1.7 tumor growth in more than 100 treated versus control mice, as observed in the cumulative tumor growth data of nine independent experiments conducted over two years. The representative tumor growth curves for individual animals within one experiment provide a sense of variability in the system.

When either monocytes or platelets are depleted from the PBMC fraction, or when plate passage is omitted, the therapeutic effect is no longer observed. The treatment is ineffective in the absence of either the immune cells or an antigen source, or in the presence of a mismatched antigen, such as YUMM1.7 tumors treated using MC38 colon carcinoma cells. For an immunizing outcome, it is also critical to avoid PBMC exposure to 8-methoxypsoralen.

FACS analysis of change of the indicated markers from corresponding IgG controls in human CD11c+cells among either freshly isolated PBMC, TI-treated PBMC, TI-treated PBMC without plate passage, depleted platelets prior to plate passage, or both of the above, showed that DC activation depends on the presence of platelets in the PBMC, and on TI plate passage. The TI-activated human DCs can effectively process and cross-present either peptide antigens, or antigens from whole 8-methoxypsoralen-exposed human tumor cells to activate human antigen-specific T cell lines in in vitro assays in a TI-and platelet-dependent manner. Specificity of T cell responses is dependent on the source of the processed antigens.

When inducing anti-cancer immunity, apoptosis of each cancer cell line must be titrated to maximize immunogenicity. Since induced immunity will be specific to immunizing antigens, detailed dissection of each system will be possible. Each experimental or clinical cancer, and each antimicrobial response, will be uniquely informative.

Each individual human cancer has its own array of tumor-specific antigens. The PhDC perform the necessary sorting and presentation of tumor antigens, without requiring their prior laboratory identification. Please keep in mind that 8-MOP and UVA light are carcinogens.

Use appropriate protective equipment and follow safety procedures when handling 8-MOP and when working with UVA light sources.