The overall goal of this procedure is to generate human regulatory T cells from primary T cells. First, peripheral blood mononuclear cells are isolated from human blood samples. CD four positive T cells are then purified from the PBMC mixture and cultured.
After six days, the cells are sorted into naive memory effector and induced regulatory T-cell subsets using a flow cytometer suppressor assays performed. Using these cells demonstrate that this method generates human regulatory T cells with suppressor activity. The main advantages of this technique over those using mouth cells is that our human method has more physiological and clinical relevance and allows for the comparison of tregs to other cell populations within a single donor.
Eliminating inner subject variability. This method can answer key questions on the regulatory cell field, such as what are the regulatory events that modulate the transition between conventional T cells and regulatory cells, or what are the signaling and transcriptional events that are specific on the regulatory cells? The implications of this technique extend toward the therapies of autoimmune diseases, inflammation conditions, and cancer because of the vital role that it regs perform in immune Homeostasis.
Generally individuals new to this technical struggle because of the variety techniques used and in some steps the narrow window for optimal cell culture conditions. Though this method can provide insight into it reg development, it can also be used to monitor the plasticity of T-cell lineage differentiation or for analyzing the capacity of T-cell milli use to induce or prevent it. Reg development Demonstrating the procedure will be Andrea McCool and the graduate students Marie Catherine Ranier Gavin Ellis, and Catalina Vale Ortega.
Begin this procedure by isolating human peripheral blood mononuclear cells from 40 to 60 liters of B Buffy coat to dilute the buffy coat, transfer it into an autoclave 500 milliliter glass bottle containing 150 milliliters sterile PBS. Then add more sterile PBS to bring the final volume up to 250 milliliters. Next, fill 10 50 milliliter conical tubes, each with 20 milliliters lympho prep solution.
Then gently overlay the Lympho prep solution with 25 milliliters of the diluted buffy coat. Being careful not to disturb the lympho prep solution. Spin the tubes for 30 minutes at room temperature at 500 times G.Make sure to turn off the centrifuge's break so as not to disturb the lymphocyte fraction.
Following the spin, the PBM CS will be at the interface between the lympho prep and the plasma medium layers to collect them. Aspirate the plasma medium off until about one milliliter still covers the buffy coat layer containing the pbmc. Use a 10 milliliter pipette to transfer pbmc to a new 50 milliliter tube.
After washing the cells twice with PBS, re suspend them in 50 milliliters of cold RPMI and count them using a hemo cytometer. Typical recovery is eight times 10 to the eighth to one times 10 to the ninth p BMCs after the concentration has been determined. Resus suspend the PBM CS at final concentration of five times 10 to the seventh per milliliter in PBS containing 2%FBS and one millimolar EDTA transfer the cells to a fresh 14 milliliter polypropylene round bottom tube.
Next, the EAs kit will be used to negatively select the cells of interest. The T-cell enrichment cocktails provided with the kit contain a tetrameric antibody complex, which has an anti dextrin antibody on one side and an antibody against unwanted cell surface antigens on the other side. When the cocktail is added to the cells, the tetrameric antibody complex connects the unwanted cells.
Dextran coated magnetic particles are then added, which bind to the tetrameric antibody complex, connecting the unwanted cell to the magnetic particle. A magnet is then applied to separate the unwanted cells from the cells of interest, which are then collected to isolate CD four positive T T-cells pipette one times 10 to the eighth to 4.25 times 10 to the eighth pbmc into a 15 milliliter conical tube. Then add 50 microliters human CD four positive T-cell enrichment cocktail of antibodies for each milliliter of pbmc.
Mix and incubate for 10 minutes At room temperature. Mix the magnetic particles to equally distribute them throughout the solution. Add 100 microliters of magnetic particles for each milliliter of pbmc.
Mix by gently pipetting two to three times and then incubate them at room temperature for 10 minutes. Add PBS containing FBS and EDTA to bring the volume up to 10 milliliters per tube mixed by gently pipetting two to three times. Next, place the uncapped tube into the cept magnet for five minutes with the tube still in the magnet.
Pour the liquid into a new 50 milliliter tube to isolate cells of interest for better recovery. Repeat this process by adding additional P-B-S-F-B-S-E-D-T-A to the tube mixing and placing the sample back in the magnet. Once the cells have been isolated, count them then centrifuge at 500 times G five minutes at room temperature.
Resuspend the cell pellet in polarization medium to reach a concentration of two times 10 to the six cells per milliliter. Then proceed to culturing as described in the next section of the video to induce regulatory T cells. Prewarm anti CD three coated six, well tissue culture plates at 37 degrees Celsius.
Once the plates are warm, aspirate the PBS from the wells. Then add four milliliters of the CD four positive enriched cell suspension to each. Well fill up any empty wells with PBS to reduce evaporation of medium incubate for three days after the incubation.
Centrifuge the plate for five minutes at 500 times G.Then without disturbing the cells on the bottom of the plate, remove half of the medium and replace with fresh medium incubate for two to three more days. The cells can now be sorted by fluorescence activated cells sorting as described in the next section or used as target cells for a suppression assay as described later on, after two to three days have passed the cells and appropriate compensation controls are prepared for fluorescence activated cells. Sorting to separate induced regulatory naive and memory T cells stain the cells to be sorted with anti CD 25 pe, anti CD 45 RA PE SCI five and anti CD 1 27 A PC as described in the accompanying document.
Also be sure to add antibodies to the appropriate compensation controls for fax analysis. After staining resuspend cells at a concentration of one times 10 to the seven cells per milliliter of washing buffer, then add 1.5 microliters of DNAs two per milliliter of cells and filter the cells through a 40 micron nylon cell strainer. Transfer the cells to five milliliter round bottom polypropylene tubes with no more than 3.5 milliliters per tube.
Resus suspend the compensation control cells in 300 microliters of washing buffer. Set the compensation on the flow cytometer to minimize cross detection by the PE A PC and PE sci-fi filters. Set gates to sort tregs naive and memory T cells into five milliliter round bottom polystyrene tubes containing one milliliter newborn calf serum to assess whether the regulatory T-cells generated during sorting are able to suppress T-cell proliferation.
A suppression assay is performed here. The nons sorted CD four positive enriched cells will function as the target cells. These should not contain CD 25 positive induced regulatory T cells.
Begin by labeling the target cells with CFSE using a cell trace kit according to the manufacturer's instructions using one microliter of five molar stock solution per milliliter of cells instead of two microliters. Next pellet a number of treg suppression inspector beads equal to the total number of cells by quick centrifugation in micro centrifuge tube. Wash the beads once with RPMI and re pellet after the spin.
Aspirate the RPMI and re suspend them to the appropriate concentration in eight microliters suppression assay medium per well to a 96 well round bottom tissue culture plate. Add CFSE stained target cells eight microliters. Inspect beads and polarized sorted cells to a final volume of fresh suppression assay.
Medium of 200 microliters. All conditions are plated in triplicate. To prepare the controls add 100 microliters of CFSE stained cells, eight microliters of inspector beads, and one times 10 to the fifth of fresh unstained cells in 92 microliters suppressor assay.
Medium per well to the first two control wells. Then add 100 microliters of CFSE stained cells and one times 10 to the fifth of fresh unstained cells in 92 microliters suppressor assay. Medium per well to the second two control wells.
Cover the plate in aluminum foil and place it in the incubator for five days in the dark. Collect the cells from each well by pipetting and place them in a five milliliter round bottom polystyrene tube. Centrifuge cells at 500 times G for five minutes at four degrees Celsius.
Aspirate medium and reus suspend them and 300 microliters cold fax washing buffer. To monitor the progress of induced regulatory T cell differentiation, purified human primary CD four positive CD 25 negative T cells were cultured in IT reg medium aliquots taken at day 0 1 3 and five were assayed by flow cytometry to assess the expression of CD 45 ra Fox P three, CTLA four, and CD 25 markers as can be seen in these pseudocolor dot plots over a five day time course cells proliferate and differentiate in vitro in response to CD three cross-linking in the presence of TGF beta and IL two, ultimately leading to a CD 45 RA negative FOX P three high CTLA four high and CD 25 high profile. This indicates the development of induced tregs from conventional T-cell precursors to assess the relative ability of the generated tregs to suppress T-cell proliferation.
CFSE labeled cells were cultured with treg suppression inspector beads in the presence of sorted naive memory and I treg cells as can be seen in this histogram. Cells CD 25 high, CD 45 RA negative CD 1 27 negative. Low induced regulatory T cells are the only subset from a five day culture that has acquired regulatory suppress ability.
This indicates that the cells generated in this system are both phenotypically and functionally suppressor regulatory T cells rather than activated conventional T cells, which may transiently express FOX P three and CD 25, but are not immunosuppressive. The presence of induced regulatory T cells completely abolishes the proliferation of CD four positive T cells. Numbers are indicative of percentage of CFSC labeled cells that have undergone division to determine that the same holds true for T-cell purified subsets.
Human primary naive and memory T-cells were stained with CFSE and cultured in it. Treg medium after five days, cells were phenotyped and the cell division rates were estimated as seen. Here tregs were induced from a naive T-cell pool and a memory T-cell pool after five day culture.
In standard IT reg medium, CFSE. Staining of initial cells shows that either naive or memory T cells differentiate to regulatory T cells only after several rounds of cell division comparative CFSE staining profiles identify the induced regulatory T cells as the most proliferative cells during the five day culture. After watching this video, you should have a good understanding on how to obtain induced TX from a population of naive memory or whole T cells obtained from a single donor While attempting this procedure.
It's important to maintain sterile technique throughout and to prevent exposure of the CFSE to the light Following this procedure. Other methods like functional assays can be performed in order to answer other questions like what are the differences in cell division rates, specific cytokine synthesis and ion channel usage between regulatory and conventional T cells After its development. This technique paid the way for researchers in the field of immunology to explore autoimmune diseases and other treg related pathologists in human patients.
Don't forget that working with human blood is extremely hazardous and precautions such as adequate blood bank testing and proper disposal should always be used while doing this procedure.
