A Vaccine-Pulsed Dendritic Cell-Based In Vitro Assay to Induce Lymphocyte Proliferation

Begin by adding the test vaccine antigens to the dendritic cell culture.

The dendritic cell recognizes the antigen, internalizes it via receptor-mediated endocytosis, fragments it into smaller peptides, and transfers the resulting antigen-MHC complex to the cell surface for presentation.

Co-culture antigen-presenting dendritic cells with naïve T lymphocytes to enable interaction between the T cell receptor and the antigen-MHC complex, along with their co-receptors.

This leads to immunological synapse formation,  activating T lymphocytes via intracellular signaling.

Activated T lymphocytes undergo clonal expansion, generating effector T lymphocytes.

Add interleukin-2, or IL-2, cytokine molecules to the culture and incubate. The binding of IL-2 triggers intracellular signaling, enabling the proliferation and survival of lymphocytes.

Take a small volume of the culture and centrifuge. Discard the supernatant. Resuspend the cell pellet with antigen-presenting dendritic cells and transfer it to the same well.

Restimulation with antigen increases lymphocyte proliferation.

Perform downstream analysis to quantify specific markers associated with lymphocyte proliferation.

To generate antigen-pulsed MoDCs, add 1 microliter per milliliter of rabies virus vaccine or RV suspension to 1 milliliter of naïve 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 1 milliliter of ice ice-cold PBS per well, and mixing it thoroughly. Transfer the suspension to a 15-milliliter tube.

Wash the wells with 2 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 500 g for 7 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 1 milliliter of the naïve lymphocyte cell suspension and 1 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 1 milliliter of the co-culture to a sterile 1.5-milliliter tube, and centrifuge the tube. Then, resuspend the cell pellet with 1 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 naïve lymphocytes and monocytes, transfer 1 milliliter of the cell suspension to a sterile 1.5-milliliter tube using a pipette, and centrifuge for 10 minutes at 500 g. Then, resuspend the pellet in 1 milliliter of PBS and transfer it to a 15-milliliter tube. Also, collect the residual cells and the corresponding tubes using 1 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 7 minutes at 850 g, 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 real-time 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.