This protocol represents a functional in vitro assay using bovine monocyte-derived dendritic cells to measure vaccine immunogenicity prior to in vivo studies. Therefore, filling an existing gap in livestock vaccinology. It is the generation and application of dendritic cells.
As the most potent antigen-presenting cells, dendritic cells have become a key research target for understanding intracellular immune mechanisms including vaccine efficacy. This assay can be implemented as a tool for screening vaccine candidates, as a quality control state for vaccine production, and for antigen and adjuvant selection. Demonstrating the procedure will be Richard Kangethe, scientist in the animal production and health laboratory.
Begin by inverting and mixing the blood obtained from the calf's jugular vein puncture with heparinized vacutainers. Then transfer 20 milliliters of heparinized blood to a sterile 50 milliliter tube and dilute it with 10 milliliters of phosphate buffered saline or PBS. Pipette 15 milliliters of lymphocyte isolation medium to a sterile 50 milliliter tube and tilt the tube to a 45 degree position.
Carefully layer 30 milliliters of blood PBS medium on top of the lymphocyte isolation using a 25 milliliter pipette and slowly return the tube to a vertical position before centrifuging it. Then collect the thin white layer of peripheral blood mononuclear cells or PBMC layer using a Pasteur pipette and transfer it into a new 50 milliliter tube. Wash the harvested PBMCs two times using 40 milliliters of PBS per wash and mix it thoroughly.
After centrifuging, resuspend the pellet in 15 milliliters of ammonium chloride potassium buffer or ACK buffer. After 10 to 15 minutes of incubation at room temperature, add up to 40 milliliters of PBS and centrifuge the tube with maximum acceleration and deceleration. Resuspend the pellet in 10 milliliters of complete culture medium.
Then add five microliters of CD14 MicroBead buffer per 10 million cells and mix thoroughly using a pipette. For flow cytometry analysis, add 10 microliters of CD14 fluorescein isothiocyanate or FITC staining antibody to the PBMCs and incubate it for 15 minutes at four to eight degrees Celsius before proceeding with the flow cytometry. To the unstained PBMCs, add one milliliter of fax buffer and centrifuge it for seven minutes at 500g and four degrees Celsius.
Then resuspend the pellet in 500 microliters of fax buffer per 100 million cells. Next, insert the immunomagnetic cell separation column into the separator and place a 50 milliliter collection tube under the column outlet for collecting the flow-through. After washing the column with one milliliter degassed fax buffer, replace the used 15 milliliter tube with a new one.
Pipette a hundred million cells in 500 microliters of buffer at a time allowing the cell suspension to pass through the column. Then rinse the column three times with three milliliters of degassed fax buffer each time. After collecting the flow-through, label the first eluted naive lymphocyte fraction as the CD14 negative cell fraction.
Remove the column from the separator and place a new sterile 15 milliliter tube under the column for the effluent collection. Add five milliliters of fax buffer into the column and immediately push it through using the plunger. Collect and label the second flow-through or the naive monocyte fraction as the CD14 positive cell fraction.
Add complete culture medium to the harvested CD14 positive monocytes to attain 1 million cells per milliliter. Next, add one milliliter cell suspension to each well of a sterile 24-well plate before supplementing each well with 40 microliters of the cytokine cocktail provided in the kit. On day two, transfer half of each well's content to individual 1.5 milliliter tubes using a pipette.
After centrifuging the 1.5 milliliter tubes at 500g for seven minutes, discard the supernatant and resuspend the pellet in 500 microliters of fresh complete culture medium. Transfer 500 microliters of this resuspended cell suspension back to the corresponding wells so that the final volume in the well becomes one milliliter. Enrich each well with 20 microliters of cytokine cocktail.
To generate antigen-pulsed MoDCs, add one microliter per milliliter of rabies virus vaccine or RV suspension to one milliliter of naive MoDC culture in the 24-well plate and incubate the plate for 48 hours at 5%carbon dioxide and 37 degrees Celsius. On day seven, keep the 24-well plate containing the antigen-pulsed MoDCs on ice for 10 minutes before adding one milliliter of ice cold PBS per well and mixing it thoroughly. Transfer the suspension to a 15 milliliter tube.
Wash the wells with two milliliters of ice cold PBS. Collect the residual cells within each well and transfer the washed contents into their respective tubes. Centrifuge the cell suspension at 500g for seven minutes.
After discarding the supernatant, resuspend the antigen-pulsed MoDCs cell pellet in a complete culture medium to adjust the final concentration of 100, 000 cells per milliliter. For MoDC-lymphocyte co-culture, seed the wells of a sterile 24-well plate with one milliliter of the naive lymphocyte cell suspension and one milliliter of antigen-pulsed or non-antigen-pulsed MoDC suspension on the seventh day of the antigen pulse. After two days of incubation, supplement each well with 20 nanograms per milliliter recombinant interleukin-2 and continue the incubation for another 120 hours.
On day 14, transfer one milliliter of the co-culture to a sterile 1.5 milliliter tube and centrifuge the tube. Then resuspend the cell pellet with one milliliter of antigen-pulsed or non-pulsed MoDCs. Mix well and transfer the cell suspensions to their corresponding wells.
After cell surface staining of the PBMCs and naive lymphocytes and monocytes, transfer one milliliter of the cell suspension to a sterile 1.5 milliliter tube using a pipette and centrifuge for 10 minutes at 500g. Then resuspend the pellet in one milliliter of PBS and transfer it to a 15 milliliter tube. Also collect the residual cells and the corresponding tubes using one milliliter of PBS.
Then add 10 milliliters of PBS to the 15 milliliter tube containing the cells and mix by pipetting. After centrifuging the tube for seven minutes at 850g, remove the supernatant and carefully resuspend the cell pellet with residual suspension left after decanting the supernatant. Transfer the cell suspension to a V-bottom 96-well plate and seal the wells to avoid spillage.
Once the staining is complete, perform the flow cytometer analysis. From the realtime preview, adjust the threshold of fluorescence including voltage and gain and cell size to eventually draw gates around the desired cell population while excluding any cellular debris. CD14 cell staining and flow cytometry showed that the naive monocyte fraction comprised 98%CD14 positive monocyte cells and was functionally capable of antigen uptake.
The naive MoDCs phenotypically characterized by assessing the expression of MHC Class 2 and co-stimulatory CD86 and CD40 cell surface markers validated the dendritic cells or DC-like phenotype. During the MoDC lymphocyte co-culture on day nine, a morphological change showing dendrite extension was observed in the RV pulse to MoDCs. Compared to the non-pulsed MoDC lymphocyte culture, a significant increase in lymphocyte proliferation was demonstrated by the upregulation of the Ki67 and CD25 activation markers on the CD4 positive and CD8 positive T-cells on day 16 in the pulsed MoDC lymphocyte co-culture.
The CD8 positive T-cells from the mature RV pulsed MoDC co-cultures exhibited an eightfold upregulation of Ki67 compared to the non-specific group. The CD4 positive cells in the same co-culture showed a sevenfold increase in Ki67 compared to controls, demonstrating the ability of RV primed MoDCs to present the RV antigen to naive lymphocytes and activating them in an in vitro condition. The qPCR quantification of co-cultures showed more than 30%increase in interferon gamma expression and more than 5%increase in Ki67 expression in all the co-cultures using GAPDH as a calibrator.
A significantly higher concentration of secreted interferon gamma was observed in the RV pulsed MoDC lymphocyte co-culture compared to the non-specific treatment group. Perform the layering step very slowly and carefully, ensuring that blood is laid on top of the lymphocyte isolation medium. Be extra careful to collect all PBMCs without collecting too much material from the other layers.
Other methods such as flow cytometry, ELISA and quantitative PCR, among others, can be applied after this procedure to evaluate the activation of immune markers.
