This novel bone marrow transplant protocol can be used to investigate how host dendritic cells regulate graft-versus-host and graft-versus-leukemia responses after transplantation. Ex vivo cell regenerations allows this protocol to be adapted to test the role of other immune cell population such as macrophage and neutrophils simply by modifying the culture condition. Demonstrating the procedure with me will be lab manager Krystal Hossack and undergraduate Sanjeev Gurshaney.
For donor T-cell-depleted C57BL/6 bone marrow preparation, harvest the femur and tibia according to standard protocols from euthanized donor mice and transfer the bones into a 92 millimeter diameter Petri dish containing 1%RPMI medium. Using scissors, remove the ends from each bone and fill a three milliliter syringe equipped with a 26 gauge needle with fresh 1%RPMI medium. Insert the needle into one end of one bone and depress the plunger to flush the bone marrow out of the cavity and into a collection cell strainer.
When all of the bone marrow has been collected, strain the bone marrow pieces through a 75 micrometer mesh filter and transfer the single-cell suspension into a new 50 milliliter tube. Sediment the cells by centrifugation and resuspend the pellet at a 20 times 10 to the six cells per milliliter concentration in PBS supplemented with 0.5%BSA. Next, add 0.05 micrograms of THI1 antibody per one times 10 to the six cells for a 30-minute incubation at four degrees Celsius.
At the end of the incubation, wash the cells twice with 10 milliliters of ice cold PBS and resuspend the pellet at a two times 10 to the seven cells per milliliter concentration in 0.5%BSA supplemented with 10%young rabbit complement and 2%DNAse in sterile water for a 45-minute incubation at 37 degrees Celsius. At the end of the incubation, wash the cells twice with 15 milliliters of fresh PBS per wash and resuspend the pellet in 20 milliliters of PBS containing 0.5%BSA. Pool the lymph nodes and the spleen in a 40 micrometer pore strainer and use a syringe plunger to macerate the tissues through the filters to release the cells.
Wash the strainer and plunger with RPMI medium supplemented with 1%FBS to collect all of the cells and sediment the cells by centrifugation. Add five milliliters of ACK lysis buffer to the cell pellet. After five minutes at room temperature, stop the reaction with five milliliters of medium supplemented with 1%FBS and pellet the white blood cells by centrifugation.
Resuspend the pellet in five milliliters of MACS buffer for counting and dilute the cells to a concentration of two times 10 to the eight cells per milliliter in fresh MACS buffer. Add the appropriate concentration of anti-erythroid, anti-granulocyte, anti-B-cell, and anti-NK-cell depletion antibodies per one times 10 to the six cells for a 15-minute incubation at four degrees Celsius. At the end of the incubation, wash the cells with 10 milliliters of ice cold MACS buffer and resuspend the pellet at a one times 10 to the eight cells per milliliter concentration in fresh MACS buffer.
Next, add 0.22 microliters of Anti-Biotin MicroBeads per one times 10 to the six cells with mixing for a 15-minute incubation at four degrees Celsius. At the end of the incubation, wash the cells with 10 milliliters of ice cold MACS buffer and collect the unbound enriched T-cells by magnetic bead separation using MACSxpress separator according to standard bead isolation protocols. At the end of the separation, sediment the T-cells by centrifugation and resuspend the pellet in five milliliters of fresh MACS buffer for counting.
To generate bone marrow-derived dendritic cells, isolate the bone marrow from the femurs of wild type or factor B knockout mice as demonstrated and lyse the red blood cells in five milliliters of ACK lysis buffer, stopping the reaction with 10 milliliters of RPMI supplemented with 1%FBS after five minutes. After collecting the whole blood cells by centrifugation, resuspend the pellet in 10 milliliters of culture medium to a two times 10 to the sixth cells per milliliter concentration and add 20 nanograms per milliliter of GM-CSF to the cells before seeding 10 milliliters of cells onto individual 100 by 15 millimeter Petri dishes at 37 degrees Celsius and 5%carbon dioxide for six days. On day six, activate the bone marrow-derived dendritic cell cultures with 25 micrograms per milliliter of LPS per plate.
At least 85%of the cultured bone marrow cells differentiate into dendritic cells. The T-cell purity after magnetic bead enrichment is 90%The major MHC mismatched C57BL/6 BALB/c model closely corresponds to GVHD development after transplantation. The peak of severity occurs approximately 11 days after cell transfer followed by a reduction in the clinical scores and body weight recovery up to day 16.
The recipients uniformly succumbed to GVHD by 30 to 40 days posttransplant. Interestingly, transplantation with factor B knockout dendritic cells improves recipient survival and GVHD clinical scores. Luciferase-transduced A20 B-cell lymphoma allows the monitoring of tumor growth in live animals.
In this representative experiment, all of the wild type BALB/c recipients that received bone marrow alone plus A20 died of a tumor relapse. Wild type BALB/c recipients transplanted with bone marrow-derived ACC1-depleted donor T-cells died of GVHD. In addition, animals that received donor bone marrow and ACC1-depleted T-cells died of both GVHD and tumor relapse.
To generate enough bone marrow cell, the collected tibia and femur bones must be completely free of muscle tissue before the bone marrow correction. Negative purification of the bone marrow cells with biotinylase anti-CD3 and Anti-Biotin MicroBeads can also per performed to deplete the lymphocyte from the bone marrow.
