The overall goal of this protocol is to develop and characterize murine tolerogenic dendritic cells for the evaluation of their immunotherapeutic utility in the treatment of autoimmune disorders, such as multiple sclerosis. This method can help answer key questions in the tolerogenic dendritic cell therapy field by providing a standardized method for developing and functionally characterizing tolerogenic dendritic cells. The main advantages of this technique are that it can be used for successful tolerogenic dendritic cell development and for functional tolerogenic dendritic cell efficacy testing.
The implications of this technique are significant. They extend toward the development of cellular immunotherapy for autoimmune disorders, as tolerogenic dendritic cells can reset an abnormal immune response while remaining subject to intrinsic immune regulatory mechanisms. After harvesting the hind leg bones from eight to 10 week old C57BL/6 mice, according to standard protocols, use surgical blades and forceps to dissect as much tissue as possible and place the clean tibias and femurs in a six centimeter culture dish containing 70%ethanol.
Trim both ends of the bones with a surgical blade and use a three milliliter syringe, equipped with a 23 gauge needle, to flush the marrow from the first bone with three milliliters of PBS into a 15 milliliter conical tube. When all of the marrow has been collected, centrifuge the cell suspension and resuspend the pellet in one milliliter of red blood cell lysis buffer. After five minutes, stop the lysis with nine milliliters of PBS and collect the cells by centrifugation.
Resuspend the white bone marrow cell pellet in 10 milliliters of culture medium and filter the cells through a 40 micrometer cell strainer into a new tube. Adjust the cells to a one times 10 to the sixth cells per milliliter concentration in culture medium supplemented with GM-CSF and IL-4. And plate three milliliters of cells into each well of a 6-well plate for a three day incubation at 37 degrees Celsius and 5%carbon dioxide.
On the third day, wash the cells in each well with two milliliters of PBS and gentle swirling to remove the non-adherent cells. Then feed the cells with three milliliters of fresh culture medium supplemented with GM-CSF and IL-4 and return the cells to the cell culture incubator, adding three more milliliters of fresh culture medium, supplemented with cytokines to each well after two days. On day seven of culture, place the plate on ice.
After 10 minutes, gently pipette the culture medium in each well to dislodge the loosely adherent, immature, bone marrow-derived dendritic cells into suspension. Then, pool the cells for collection by centrifugation and resuspend the pellet in the appropriate volume of fresh culture medium for subsequent downstream analysis. To measure syngeneic T cell proliferation, first use the back end of a three milliliter syringe plunger to mash a spleen from an eight to 10 week old OT2 C57BL/6 mouse through a 40 micrometer cell strainer and rinse the strainer with PBS.
Pellet the pooled cell suspension by centrifugation and resuspend the pellet in 400 microliters of magnetic cell sorting buffer and 100 microliters of CD4 positive T cell biotin-antibody cocktail at four degrees Celsius for five minutes. At the end of the incubation, add 300 microliters of sorting buffer and 200 microliters of anti-biotin beads to the cells for a 10 minute, four degrees Celsius incubation and place a magnetic bead column and a pre-separation filter into an appropriately-sized cell separation magnet. Rinse the column with three milliliters of sorting buffer and add nine milliliters of sorting buffer to the cells.
After centrifugation, resuspend the cell and bead pellet in three milliliters of sorting buffer and add the cell suspension to the column to collect the CD4 positive T cell eluate in an appropriate container. Then wash the column with another three milliliters of sorting buffer, collecting the flow-through and place the T cells on ice. For syngeneic splenic pan dendritic cell isolation, place the spleen from an eight to ten week old C57BL/6 mouse in a six centimeter culture dish and use a one milliliter syringe, equipped with a 25 gauge needle, to inject one milliliter of Collagenase D solution into the spleen two times.
Next, use scissors to cut the spleen into small pieces and incubate the pieces with shaking at room temperature for 25 minutes. At the end of the incubation, add 500 microliters of 0.5 molar EDTA to the tissue slurry for a final five minute incubation at room temperature. At the end of the incubation, use the back end of a three milliliter syringe plunger to mash the spleen slurry through a 40 micrometer cell strainer and collect the cells by centrifugation.
Resuspend the pellet in 350 microliters of magnetic cell sorting buffer, 50 microliters of Fc receptor blocking reagent, and 100 microliters of pan dendritic cell biotin-antibody cocktail for a 10 minute incubation at four degrees Celsius. At the end of the incubation, wash the cells in nine milliliters of sorting buffer and resuspend the pellet in 800 microliters of fresh sorting buffer with 200 microliters of anti-biotin beads at four degrees Celsius for 10 minutes for isolation of the splenic pan dendritic cell population by magnetic bead sorting, as just demonstrated. Resuspend the cells at a two times 10 to the fifth dendritic cells per milliliter concentration and treat them with the appropriate experimental concentration of the triterpenoid of interest for one hour at 37 degrees Celsius.
During the last third of the incubation, resuspend the T cells at a one times 10 to the seventh cells per milliliter concentration in one micromolar CFSE for 15 minutes at 37 degrees Celsius. Then wash the cells with PBS and readjust the volume to a final concentration of two times 10 to the sixth T cells per milliliter. Co-culture 100 microliters of the treated dendritic cells with 100 microliters of the CFSE-labeled CD4 positive T cells in each well of a 96-well plate and add 100 nanograms per milliliter of OVA peptide 323-329 to the cells, measuring the CFSE intensity of the T cells by flow cytometry after two to three days of incubation.
Bone marrow progenitor cells cultured in complete medium, in the presence of GM-CSF and IL-4, exhibit an immature dendritic cell morphology after six days in culture. Analysis of immature day seven bone marrow-derived dendritic cells reveals the robust expression of CD11c, a specific murine dendritic cell marker, by the majority of the cultured cells. Despite a lack of effect on the expression of LPS-induced maturation markers, bone marrow-derived dendritic cells display a tolerogenic dendritic cell profile in response to CDDO-DFPA treatment as evidenced by significant reduction in pro-inflammatory gene expression and an enhanced anti-inflammatory cytokine gene expression, even after LPS stimulation.
In addition, a significant reduction in syngeneic OVA-specific T cell proliferation is observed in in vitro co-cultures in which OVA is presented by CDDO-DFPA treated dendritic cells and in vivo as evidenced by a delayed progression to experimental auto-immune encephalomyelitis after the injection of MOG-pulsed CDDO-DFPA treated dendritic cells, further confirming the tolerogenic phenotype of these cells. Once this technique has been mastered, tolerogenic dendritic cells can be developed within eight days, if the experiments are performed properly. While attempting this procedure, it's important to remember that tolerogenic dendritic cells are not homogeneous.
Their cellular phenotype depends upon the nature of the agents used for the induction of their tolerance. Following this procedure, other methods, such as detailed flow cytometric analysis, can be performed to answer additional questions about tolerogenic dendritic cell-induced antigenic-specific T cell energy or apoptosis or the ability of T cell differentiation. After watching this video, you should have a good understanding of how to develop functionally-active tolerogenic dendritic cells using known agents or how to test the ability of new substances to induce these cells.
