This study investigates the activation and proliferation of T cells through receptor-ligand interactions with antigen-presenting cells (APCs). The methodology involves culturing human peripheral blood mononuclear cells (PBMCs) and utilizing specific antibodies and cytokines to promote T cell responses.
Multiple cell-surface receptor-ligand interactions between naïve T cells and antigen-presenting cells, APCs, elicit T cell activation and proliferation.
The two primary events in this process involve the recognition of the APC-expressed antigen by the CD3-T-cell receptor complex, followed by the interaction between the APC-expressed B7 ligand and the T cell receptor CD28.
To study T cell activation and proliferation in vitro, take human peripheral blood mononuclear cells, PBMCs, suspended in a suitable culture medium. Add magnetic beads coated with anti-CD3 and anti-CD28 antibodies, and divide the cells into two conical tubes.
Add the cytokine interleukin-2, IL-2, to the first tube and interleukin-15, IL-15, to the other. Transfer the cells to a multi-well plate. Incubate.
The beads' anti-CD3 and anti-CD28 antibodies bind to the CD3-T-cell receptor complex and CD28 receptor expressed on the T cells, respectively, causing T cell activation and proliferation.
With IL-2, the cytokine binds to its receptor on the activated T cell surface, leading to its differentiation and proliferation into effector T cells. Conversely, IL-15 primarily drives T cell differentiation and proliferation into memory T cells.
Resuspend and transfer half of the cells into new wells to allow cellular growth. Add fresh medium containing IL-2 or IL-15 to maintain the cytokine-differentiated T cell populations for subsequent analysis.
To begin, wash CD3/CD28 beads by transferring 12.5 microliters of beads per million cells to a microcentrifuge tube, and adding 12.5 microliters of PBS per 12.5 microliters of the beads in the tube. Then, place the microcentrifuge tube on a suitable magnet for 1 minute, discard the buffer, and resuspend the beads in the original volume of T-cell medium.
Next, add 12.5 microliters of beads per million cells at a 1 to 2 beads to cells ratio, and divide the cells into two conditions with around 5 million cells in each. Then, add the correct volume of cytokines to each condition, and transfer the cells to multi-well plates. Incubate the cells for three days at 37 degrees Celsius and 5% carbon dioxide.
After three days of incubation, prepare fresh T-cell medium with double the concentration of cytokines. Resuspend and split the cells by transferring half the volume from each well into a new well. Then, add the same volume of freshly-prepared T-cell medium to each well.
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