This protocol can evaluate how targeted gene delivery affects B-1a cell localization and function and can be a useful proof of concept approach for examining gene therapeutic potentials in vivo. This method provides a stable and relatively efficient gene delivery to primary B-1a cells and allows the identification of donor cell phenotype and function post-adoptive transfer. This method can be used to examine diverse donor and host cell processes in vivo including cell survival, proliferation, and function, and can be applied to other adoptive transfer systems.
Peritoneal fluid harvesting can be tricky. Be sure to thoroughly disengage the cells to maximize the recovery and to avoid puncturing the intestines which can contaminate your peritoneal cell population. For peritoneal B-1 cell collection, use straight surgical scissors to make a superficial cut in the abdomen of a 12 to 14-week-old, male, CD45.1+apolipoprotein E knockout mouse, and use curved scissors to peel back the skin to expose the peritoneal wall.
Using a 10 milliliter syringe equipped with a 25 gauge needle, flush the peritoneal cavity with 10 milliliters of 37 degree Celsius RPMI-1640 medium. And grasp the base of the tail to allow the mouse to be thoroughly shaken from side to side for 15 to 20 seconds. After shaking, use the syringe to aspirate the fluid from the lower right side of the peritoneum just above the level of the hip, near the intestines, taking care to avoid disrupting the epidydimal fat depots and underlying organs.
After collecting six to seven milliliters of lavage, dispense the fluid into a 50 milliliter conical tube on ice. Use forceps to grasp the peritoneal wall above the diaphragm to allow vertical elevation of the animal so that any remaining fluid remains at the bottom of the peritoneal cavity. Use surgical scissors to make a small cut in the peritoneal wall above the liver without cutting the liver itself.
And use a glass pipet and bulb to collect any remaining peritoneal fluid. When all of the fluid has been collected, aliquot the peritoneal washout cells from all of the mice and add up to one times 10 to the eighth cells per tube concentration in 50 milliliter tubes on ice. And sediment the cells by centrifugation.
Resuspend each pellet in one milliliter of anti-CD16/CD32 antibody diluted at a one to 50 ratio in assay buffer for a 10 minute incubation at four degree Celsius. At the end of the incubation, add an equal volume of biotinylated antibody master mix to each tube for a 20 minute incubation at four degree Celsius followed by a wash in five milliliters of assay buffer per tube. Resuspend the pellets with the appropriate volume of anti-biotin microbeads and appropriate incubation duration as per the cell concentration and manufacturer's recommendation followed by a five milliliter assay buffer wash per tube.
Resuspend each pellet in 500 microliters of assay buffer and prime the appropriate number of magnetic selection columns with three milliliters of assay buffer per column. Transfer the cells onto primed columns collecting the eluent containing enriched B-1 cells in a 15 milliliter conical tube on ice. Then wash each magnetic selection column with additional assay buffer until the overall collected volume is 10 milliliters and resuspend the post-purified cell fraction at one times 10 to the sixth cells per milliliter concentration in B cell culture medium.
To stimulate the peritoneal B-1 cells set aside at least one times 10 to the seventh cells for a non-transduced control and split the remaining cells into two equal volumes for transduction. Dilute the cells to 1.5 times 10 to the fifth cells per 150 microliters of medium concentration and add 150 microliters of cells to wells of one 96-well round-bottom plate for transduction condition. Then add 100 nanomolar of TLR9 agonist to each well and place the plates in the cell culture incubator for 16 to 18 hours.
For retroviral transduction of the peritoneal B-cells thaw calcium phosphate transfection retroviral particle stocks on ice and immediately add to the control and the chemokine receptor retroviral supernatants to each well of the appropriate plate and multiplicity of infection of 20 to one in the presence of 8 micrograms per milliliter of polybrene and fresh beta-mercaptoethanol at 55 micromolar final concentration. When all of the viruses have been added, spinfect the cells by centrifugation and place the plates in the cell culture incubator for three hours. At the end of the incubation, harvest the cells for replating in fresh B cell medium and an overnight incubation.
After magnetic depletion of other peritoneal cell types, live singlet cells in the post-depletion fraction have a greater proportion of CD19+B cells compared to F4/80+macrophages, and a lack of CD5hi CD19-T cells, and they contain an increased frequency of CD19+CD5 medium B-1a cells compared to the pre-depletion fraction. And increase in frequency of successfully transduced GFP+B-2, B-1, B-1a, and B-1b cell subsets is directly correlated to the increase in virus multiplicity of infection. And the transduction efficiency can be further increased using 96 well round-bottom plates compared to the use of 24-well or 6-well plates.
CXCR4-GFP retrovirus transduction can induce a successful B-1 cell CXCR4 overexpression and increase in in vitro migration towards CXCL12 without a significant impact on B cell viability. In addition, adoptively transferred CD45.1+donor cells sustained their CXCR4 overexpression after their recovery from the bone marrow and spleens of CD45.2 recipient mice 17 weeks post-cell transfer. Of note, a positive association between the CXCR4 expression and donor cell localization to the bone marrow but not spleen has been determined.
With a positive association observed between donor cell numbers in the bone marrow and the plasma amount of anti-MDA-low density lipoprotein IgM. Many additional techniques can be utilized to answer questions such as, what is the impact of targeted gene delivery on disease development or which donor derives secreted factors impact host physiology.
