The overall goal of this procedure is to evaluate the phenotype and suppressive function of natural CD four Positive regulatory T cells expanded from the peripheral blood of H HIV V one positive individuals. This is accomplished by first isolating the regulatory T cells by density, gradient, and flow-based cell sorting. In the second step, the regulatory T cells are expanded in culture.
Next, the phenotype of the expanded cells is characterized by flow cytometry in the final step, the function of the expanded TRE population is assessed by a T-cell suppression assay. Investigating natural CD four positive TRE in HIV infected individuals has traditionally been challenging and in large part because of the scarcity of the population. The advantage of our technique is that it allows for the exvivo generation of large numbers of these very rare cells.
The method demonstrated here may help to answer key questions in the HIV immunopathogenesis field by facilitating the characterization of the function and specificity of regulatory T cells in HIV disease progression. Though this method can provide insight into peripheral blood regulatory T cells, it can also be applied to other body compartments, such as expansion of regulatory T cells from the gut associated lymphoid tissue. A major site of HIV replication Begin by carefully transferring the blood from the A CD collection tubes into 50 milliliter conical tubes for a final volume of 15 milliliters of blood per tube.
Then add 25 microliters of rosette ep, human CD four positive T-cell enrichment cocktail per milliliter of blood. Mix carefully and incubate the cell suspensions for 20 minutes at room temperature. After the incubation, carefully mix 15 milliliters of PBS supplemented with 2%FBS with the blood.
Then layer the diluted blood sample on top of 15 milliliters of histo pack in a 15 milliliter conical tube. Spin the blood for 20 minutes at 1200 Gs and room temperature with a slow start and no breaks, and then transfer the CD four positive T-cell enriched PBMC layer into a new 50 milliliter conical tube. Wash the cells in PBS supplemented with FBS.
Count the cells and then after another wash, resuspend the pellet at about 20 times 10 to the six cells per 200 microliters. Add the following antibodies at the indicated concentrations and then incubate the cells for 30 minutes in the dark at four degrees Celsius. After washing away the unbound antibody, we suspend the cells at 20 times 10 to the six cells per milliliter in additional PBS plus FBS and filter the cell suspension through a 35 micrometer nylon mesh.
Now using a cell sorter equipped for handling biohazardous material sort the CD three positive CD four positive CD 25 positive CD 1 27 low treg in ex vivo 15 media. Using this gating strategy, conventional CD three positive CD four positive CD 25 negative CD 1 27 positive T cells can then be isolated and expanded as negative controls. After sorting, wash the T-Rex cells with ex vivo 15 media and resuspend the pellet at 250 times 10 to the three cells per milliliter.
In ex vivo 15 media supplemented with human serum penicillin and streptomycin. Then after washing the human T activator CD three, CD 28 beads, according to the manufacturer's instructions, add the beads to the isolated T-Rex cells at a ratio of one-to-one bead per cell. Then after two days of culture, double the media volume and add IL two to the cells.
Now culture, the T-Rex cells for two weeks, changing the media on days 5, 7, 9, and 12. On day nine, add beads to the culture at a one-to-one ratio at the end of the culture expansion. Wash the harvested conventional and regulatory T cells in PBS.
Then use the live dead fixable violet dead cell stain kit. According to the manufacturer's protocol to stain the dead cells, wash away the excess stain in PBS and FBS. Next, add the following antibodies at the indicated concentrations and incubate the cells at 30 minutes in the dark at four degrees Celsius.
After another wash, stain the cells with a Fox P three transcription factor staining buffer set according to the manufacturer's instructions. Then add the following antibodies at the indicated concentrations and acquire the data on a flow cytometer. Also at the end of the expansion culture thaw autologous cryopreserved, ex vivo pbmc.
Rest the cells for about three hours in a 37 degree Celsius incubator in complete media. After staining dead cells as just demonstrated, wash the cells in PBS with FBS and then incubate the cells with anti CD three, PE size seven for 30 minutes in the dark at four degrees Celsius. After washing the cells.
Again, re suspend the pellet in PBS and FBS and filter the cell suspension through 35 micrometer nylon mesh then use a cell sorter equipped for handling biohazardous material to sort the viable CD three positive T-cells into R 10 media. After sorting labeled the cells with CFSE, according to the manufacturer's instructions and resuspend the cells in r plus media supplemented with human serum at one times 10 of the six cells per milliliter. Now harvest the expanded T-Rex cells and Resus, suspend them at 0.5 times 10 to the six cells per milliliter in human R 10 media.
Then further split the cell culture into 0.2, five times 10 to the six cells per milliliter and 0.125 times 10 to the six cells per milliliter dilution. Next Resus suspend anti CD two, anti CD three, anti CD 28 microbeads prepared according to the manufacturer's instructions at 0.75 times 10 of the six beads per milliliter in human R 10 media. Then further dilute the beads with more R 10 media to three different final concentrations of 0.6 2 5, 0 0.562, and 0.5 times 10 of the six beads per milliliter respectively.
Culture, the diluted cell and bead suspensions in a 96 well round bottom plate according to this protocol. After four days of culture, wash the cells and then incubate them for 30 minutes at four degrees Celsius with these antibodies. Finally acquire the data on a flow cytometer.
This first figure represents the gating strategy used to flow sort single CD three positive, CD four positive, CD 25 positive CD 1 27, low TREGS from PBMC, isolated from an HIV V one positive individual, the anti CD 25 and anti CD 1 27 antibody clones and their conjugates are crucial for good separation of the treg population. In this representative example, the treg expansion reached a 765 fold change after 14 days of culture. The expansion fold change can vary from one individual to another and can range from 100 times to 2, 500 times above the starting population.
In this figure, the gray histograms represent staining for selected treg markers. For example, CTLA four, Fox, P three, and Helios. A fundamental characteristic of regulatory T cells is their capacity to suppress T cell effector function using a standardized flow-based T cell proliferation assay.
This figure shows how regulatory TT cells can suppress the proliferation of activated T-cells at different T-cell to treg ratios. After watching this video, you should have a good understanding of how to isolate and expand functional tregs from HIV positive individuals. While attempting this procedure, it's important to remember to apply stringent gating criteria for sorting of the T-Rex as conventional T-cells will also be expanded and may contaminate and overgrow the cell culture Following this procedure.
Other studies like comparing the function or gene expression of fluorescent HIV in vitro infected versus uninfected tregs can be performed. Don't forget that working with HIV positive specimens can be hazardous in that precautions such as following strict bloodborne pathogen standard operating procedures and working with a sales order equipped to handle biohazardous material should always be taken while performing this procedure.
