In Vitro Differentiation of Human CD4⁺FOXP3⁺ Induced Regulatory T Cells (iTregs) from Naïve CD4⁺ T Cells Using a TGF-β-containing Protocol

The overall goals of this experiment are to induce human FOXP3-positive regulatory T cells from naive CD4 T cells in vitro, and to analyze their phenotype. This method can help to answer key questions in the T cell immunology field, such as how does regulatory T cell differentiation occur on the molecular level? The main advantage of this technique is that it allows the reproducible generation and phenotyping of human FOXP3-positive regulatory T cells.

Although this method can provide insights into regulatory T cell differentiation, it can also be modified to study other CD4 T cell subsets and other immune cells of interest. The visual demonstration of this method is crucial because the PBMC and magnetic bit isolation steps are easy to understand when shown rather than described. To isolate peripheral blood T cells, begin by adding 15 milliliters of room temperature density gradient medium into five 50-milliliter tubes per buffy coat, and bring the final buffy coat volume up to 180 milliliters with room temperature PBS.

Next, tilt a tube of density gradient medium to the side and slowly layer approximately 35 milliliters of diluted blood onto the density gradient medium without mixing the solutions. Separate the cells by centrifugation. Then, transfer the white PBMC layers between the density gradient and the plasma phases from each tube into new 50-milliliter conical tubes.

Wash the PBMC with 200 milliliters of fresh PBS and re-suspend the pellets in complete medium for depletion of the peripheral blood monocytes by plastic adherence. After collecting the floating cells, wash the PBMC three times in up to 50 milliliters of fresh PBS, re-suspending the final pellet in 40 microliters of four degrees Celsius isolation buffer and 10 microliters of naive CD4-positive T cell biotin antibody cocktail per one times 10 to the seventh cells with thorough mixing. Incubate the cells for 10 minutes at four degrees Celsius, then wash the cells with 40 milliliters of four degrees Celsius PBS and re-suspend the pellet in 80 microliters of isolation buffer and 20 microliters of anti-biotin microbeads per one times 10 to the seventh cells for a 15-minute incubation at four degrees Celsius.

While the cells are incubating, place an LS column onto a magnet, and equilibrate with three milliliters of isolation buffer, then wash the cells in 50 milliliters of PBS, and re-suspend the pellet in three milliliters of fresh isolation buffer. Now add the cells to the column, collecting the naive CD4-positive T cell-containing flowthrough in a 15-milliliter tube. When the column stops dripping, rinse the tube with two milliliters of isolation buffer and transfer the wash to the column.

After the last wash, pellet the magnetically sorted T cells by centrifugation, followed by two washes in 15 milliliters of medium, resulting in a greater than 94%pure naive CD4-positive T cell population. To induce regulatory T cells, add 50 microliters of the appropriate stimuli to the corresponding wells of an anti-CD3-coated 96-well plate. Then fill the empty wells, including the margin wells, with 200 microliters of PBS, and prewarm the plates at 37 degrees Celsius and 5%carbon dioxide.

Adjust the concentration of the resting naive T cells to a 2.2 to 2.6 times 10 to the sixth cells per milliliter density in 37 degrees Celsius medium, and add 50 microliters of cells to each well. When all of the cells have been seeded, wrap the plate in aluminum foil for protection from light, and return the plate to the cell culture incubator. After two to three days, small clusters of proliferating cells that emerge into bigger clusters at later time points should become visible by light microscopy.

After four to six days, the medium changes from slightly orange to yellowish, and the clustered cell pellets are visible to the naked eye. To evaluate the cell surface marker expression, use a pipette to re-suspend the cells in each well, and transfer the cells into a new 96-well U-bottom plate, leaving one well free around each well to prevent spillover contamination during the standing process. When all of the cells have been transferred, centrifuge the plate and discard the supernatant.

Leaving the plate upside down on an absorbent paper, tap the plate one time, and immediately turn it right side up. Vortex to re-suspend the cells in the remaining liquid. Then add 25 microliters of surface-standing premix to each well, and incubate the plate for 30 minutes at four degrees Celsius in the dark.

At the end of the incubation, wash the wells two times in 200 microliters of PBS, removing the supernatant, and re-suspending the cells after each wash, as just demonstrated. Then perform viability and intracellular staining for FOXP3 and other markers of interest. In these graphs, the flow cytometry gating strategy for CD25-positive CD4-positive FOXP3-positive regulatory T cells is shown.

Upon in vitro stimulation, most of the cells up regulate CD25, the expression of which is reduced in the presence of rapamycin. Only under the addition of inducible Treg factors does a clear population of FOXP3-positive cells with intracellular protein expression levels as high as a naturally occurring Tregs become apparent. Indeed, each induced Treg condition demonstrates a specific and reproducible pattern of cell proliferation.

With TGF beta inducing a slight increase in proliferation, and all-trans retinoic acid increasing T cell proliferation dramatically, while the addition of rapamycin to the cultures strongly reduces the cell cluster size. QRT-PCR analysis of induced Tregs indicates that FOXP3 mRNA expression is higher in all of the induced Treg cultures compared to control-stimulated cells, with rapamycin-treated induced Tregs exhibiting the lowest levels of FOXP3 mRNA of the induced Treg cultures. Further, FOXP3 mRNA expression in naturally occurring Tregs is observed to be higher than that exhibited by any of the inducible regulatory T cell populations.

This technique paves the way for researchers in the T cell immunology field to explore the molecular mechanisms of Treg generation and function in healthy donors or in patients with autoimmune disease or cancer. After watching this video, you should have a good understanding of how to isolate human naive CD4-positive T cells and how to differentiate and phenotype the resulting NUs to regulatory T cells. Please don’t forget that working with human blood or paraformaldehyde can be extremely dangerous.

So precautions such as pre-process, do all vaccinations, wearing protective clothing, and using proper waste disposal, should be taken into consideration while performing these procedures.