The overall goal of this procedure is to identify, isolate, and clone human T cells with a defined allo antigen specificity. This is accomplished by first isolating T lymphocytes from whole peripheral blood by density gradient centrifugation. In the second step, the allo antigen specific T cells are labeled with Fluor four conjugated peptide, HLA, tetramers and enriched by magnetic separation.
Next, the labeled T cells are isolated by single cell flow cytometry cell sorting. Finally, the single cell cultures are stimulated with anti CD three and anti CD 25 antibodies. Ultimately, the allo reactivity of the antigen specific T cells can be evaluated by flow cytometric analysis.
The main advantage of this technique over other techniques such as bulk lymphocyte stimulation and limiting dilution is that this technique enables the generation of T-cell clones with specific reactivity to well-defined allo antigens. Generally, individuals who are new to this method may have difficulty in culturing and expansion of the T-cell clones. This requires careful daily monitoring of the cultures Before beginning the procedure.
Aliquot four milliliters of room temperature density gradient into two to four sterile 15 milliliter conical centrifuge tubes use 70%ethanol to wipe the outside of one to two filled green top blood collection tubes containing blood from a healthy volunteer and then carefully remove the tops, decant the blood into a sterile 50 milliliter centrifuge tube, and then res each collection tube with 10 milliliters of PBS. Pool the washes with the decanted whole blood and mix a solution gently then incubate the cells with 25 microliters of human T-cell enrichment cocktail per two milliliter total volume of the diluted blood. After 20 minutes, use a 10 milliliter pipette to layer up to 10 milliliters of the blood gently on top of the density gradient medium, taking care not to disrupt the surface of the gradient.
Centrifuge the cells for 10 minutes and then use a five milliliter pipette to carefully transfer the leukocyte layer from the interface of the density gradient and the diluted plasma into a sterile 50 milliliter conical tube. Bring the volume of the collected peripheral blood lymphocytes up to 50 milliliters with PBS and gently mix the cell suspension. Then count the cells and remove a one milliliter aliquot for analysis.
Next, after spinning down the cells resus, suspend the pellets in one milliliter of flow cytometry buffer and mix 50 microliters of the diluted allo antigen peptide MHC tetramer into the cell suspension. Transfer the cells into a sterile five milliliter tube, and then after 30 minutes, wash the cells in two milliliters of sort buffer resus. Suspend the palate in 100 microliters of sort buffer, and then incubate the cells in 10 microliters of biotin selection cocktail for 15 minutes.
Next, incubate the cells in five microliters of magnetic particles for 10 minutes, and then gently mix the cell suspension with 2.5 milliliters of sort buffer. Remove a 100 microliter aliquot of analysis of preen enrichment cells and then place the tube into the cell separation magnet. After five minutes, hold the tube and magnet together and decant the contents into a fresh five milliliter tube.
Then remove the tube from the magnet and wash the cells in two milliliters of sort buffer resuspend the pellet in cold sort buffer and aliquot the cells in two milliliters of cold sort buffer per sample type. After spinning down the samples again, re suspend the pellets in 25 microliters of cold sort buffer and incubate the cells in five microliters of human FC block on ice for 20 minutes. Next, incubate the tubes with 20 microliters of the appropriate antibody cocktail.
After 20 minutes, wash the cells in two more milliliters of cold sort buffer and resuspend the pellets at one to 2 million cells per milliliter. In more buffer then aliquot 100 microliters of human T cell culture medium into each well of a 96 well round bottom cell culture plate and store the plate on ice until the sorting has completed. After sorting, centrifuge the plate and then place the cells at 37 degrees Celsius in a 6%CO2 incubator.
Next, mix the appropriate volume of activator beads by vortexing and transfer the appropriate volume of beads into a sterile five milliliter tube. After washing the beads on the magnet, decant the wash solution and add 100 microliters of human T-cell culture, medium per 0.5 microliters of microbeads to the tube. Then aliquot 100 microliters of the activator bead suspension to each well of the 96 well plate and return the cells to the incubator.
Small clusters of proliferating cells may be observed microscopically after five to seven days of culture, 14 days after cell isolation. Carefully replace 100 microliters of media from the top of the cultures with 100 microliters of fresh human T-cell culture. Medium large cell clusters should be visible microscopically two to three days after the feeding macroscopically cell pellets should become visible during the 14 days following the medium change.
After 28 days following cell isolation, transfer the entire 200 microliter volume of the growth positive cultures to individual wells of a 48 well tissue culture plate containing 200 microliters of human T-cell culture medium. Then add 12.5 microliters of the stimulatory anti CD three, anti CD 28 microbeads in 100 microliters of human T-cell culture medium, and return the plate to the incubator three to five days after the restimulation. The medium should begin turning straw yellow.
At this point, transfer the cultures to individual wells of a 24 well tissue culture plate and add 500 microliters of human T-cell medium to each culture. Finally, at 10 to 14 days following restimulation, collect 200 microliters of each T-cell culture to assess their allo antigen specificity by flow cytometry analysis of their peptide. MHC Tetramer binding The expected yield of allo antigen specific T cells will depend on the donor the allo antigen used and the corresponding frequency of reactive T cells in the donor.
For example, an average healthy donor will have 4, 500, 000 to 10 million leukocytes per milliliter of blood, approximately 20%of which are T lymphocytes. These dot plots illustrate the results of measuring the binding of T cells from an HLAD four negative donor to a specific allo antigen. For example, in this experiment, a frequency of one in 4, 776 allo antigen specific T cells were enumerated from a starting number of 20 million leukocytes from this starting cell population.
88 individual temer labeled T cells were able to be isolated then after two rounds of microbead stimulation and culture, two growth positive cultures were identified by a microscopic examination and their allo antigen specificity was assessed by examining their binding of the A 2 30 48 HLA DR.Four tetramer. While attempting this procedure, it's important to remember to monitor the cell cultures daily to prevent cell death and overgrowth of the cultures. Following this procedure, the T-cell clones can be used to answer additional questions such as examining the biochemistry of allogeneic ligand recognition and how that translates into effective immune responses.
