The overall goal of this procedure is to isolate, grow, and analyze HPV antigen specific T-cell clones from patients. First, establish a CD eight T-cell line, perform an SBOT assay and estimate the frequency of antigen-specific T-cell. After isolating the antigen-specific T-cell clones on the basis of interferon gamma secretion, perform a high throughput screening interferon gamma sbot assay to identify epitope specific T-cell clones.
Continue to characterize the T-cell clones for the minimal optimal peptide. Epitope sequences also determine whether the epitope is endogenously processed. Identify the HLA restriction element and assess cross reactivities to other high risk HPV types.
Ultimately, the goal is to describe novel and clinically relevant HPV T-cell epitopes through the screening and characterization of antigen-specific T cells from specific T-cells from patients. The main advantage of this technique of exiting methods such as using LY assay to identify antigen specific T-cell is that the likelihood of success is much higher. This method can help answer key questions in the immunology field, such as which portions of HPV do T cells recognize The implications of this technique extend toward therapy or diagnosis of cervical cancer because vaccines and immunotherapies can be developed based on the knowledge about T-cell specificities.
This method can provide insight into CD eight T-cell epitopes and it can also be applied to other systems such as CD four T cell epitopes, an interferon gamma SPO assay with a CD eight T cell line established from subject one, determined that the H PV six 16 E 6 46 to 70 region contained a T cell epitope. These epitope specific T cells were selected on the basis of interferon gamma secretion. Prepare the feeder cell mixture of irradiated allogenic peripheral blood mononuclear cells, be lymphoblasts cells and phyto hemagglutinin in complete E cells medium.
Now using serial dilution of the isolated epitope specific T cells in the feeder cell mixture, prepare a concentration of five cells per milliliter with a multichannel pipetter plate. 100 microliters per well of diluted epitope specific T cells into 96 well round bottom plates. Culture for five days at 37 degrees Celsius and 5%CO2 stimulate cells with recombinant human interleukin two on day 11 to 14.
Visually identify growing T-cell clones as large pellets at the bottom of the plate wells. Sometimes the media turns yellow. Prepare a new feeder cell mixture and complete ecel, medium aliquot one milliliter per well of a 24 well plate.
A successful experiment hinges on being able to isolate and grow T-cell clones with the 200 microliter micro pipetter. Transfer the T-cell clones individually to separate wells of the pleated feeder cells. Repeat until all T-cell clones are transferred.
Coat wells of sterile sbot plates with 50 microliters of five microgram per milliliter. Primary anti interferon gamma monoclonal antibody after one hour incubation at four degrees Celsius. Wash three times with 200 microliters of PBS.
Then block non-specific binding with 50 microliters of 5%pooled human serum in RPMI 1640. Always disperse any large bubbles by popping with a sterile needle. Incubate for an hour at 37 degrees Celsius and 5%CO2.
Next, add one times 10 to the fifth autologous LCLs in 50 microliters of RPMI 1640 with 5%human serum. Proceed to mix cells of each T-cell clone with a one milliliter pipette and transfer a hundred microliters to a sterile einor tube. Dilute with 500 microliters of RPMI 1640 containing 5%pooled human serum.
Then pellet cells by centrifugation, decant the supernatant and resuspend cells in 110 microliters of RPMI 1640 containing 5%human serum. Now plate 50 microliters of each clonal cell suspension for rapid screening in identically positioned wells of the two Ellis spot plates for the PHA positive control wells. Combine remaining media from several einor tubes and plate 50 microliters each to the next identically positioned wells.
Also prepare negative control wells with 50 microliters of media. Now for the test plate, add 50 microliters of peptides contained in the positive region to the positive control wells. Add 10 microliters of PHA stock solution and 40 microliters of RPMI 1640 and to the media only plate.
Add 50 microliters of RPMI 1640 with 5%pooled human serum incubate the Ellis spot plates overnight at 37 degrees Celsius 5%CO2. Do not move the plates during the entire incubation period. Decant media, then wash wells three times with 200 microliters of PBS containing 0.05%tween 20.
Next, add 50 microliters of the biotin conjugated anti interferon gamma monoclonal antibody and incubate for two hours. Wash three times with 200 microliters of PBS containing 0.1%tween 20. Then add 50 microliters of freshly prepared avid and bound biotinylated horse radish peroxidase each incubate plates at 37 degrees Celsius at 5%CO2 for one hour after three washes with PBS containing 0.1%tween 20, add 50 microliters stable.
Dino benzine allow color development for five minutes at room temperature. Now wash three times with 200 microliters of deionized water. Allow the plate to dry completely.
Count the spots using an automated Ellis spot reader. Select T-cell clones for epitope specific confirmation. Then perform the Ellis spot assay with 10 to the third clonal T cells for reactivity to one each of the three peptides contained in the epitope region.
Prepare autologous LCLs infected with wild type recombinant vaccinia virus and also autologous LCLs infected with recombinant vaccinia virus expressing the HPV 16 E six or E seven protein. Again, use the Ellis bot assay on selected T clonal cells to compare reactivity to autologous LCLs infected with wild type recombinant vaccinia virus and reactivity to autologous LCLs infected with recombinant vaccinia virus, expressing the HPV 16 E six or E seven protein with select T-cell clones. First, perform an Ellis Spott assay using a series of overlapping nine MER peptides covering the epitope region.
Then perform an LY spot assay for reactivity to a series of peptides of varying length. Proceed to a lie spot assay with a concentration range of few peptides. Most likely to contain the minimum and optimal amino acid sequence.
Prepare allogenic LCLs isolated from the human subject of interest sharing one or two HLA class one molecules. Select well brewing T-cell clones. Perform an Ellie spot assay using 10 to the fifth of the allogenic LCLs 10 to the third T-cell clone cells, and with the minimal and optimal peptide at 10 micromolar.
Once the epitope specific T cell clones have been characterized, they can be tested for cross reactivity to other high risk HPV types. Simply screen select clonal T cells of interest with an Ellie spot assay for reactivity with peptides containing homologous amino acid sequences from the other high risk HPV types. Subject one is a 22-year-old African American woman with a recent history of high grade squamous intraepithelial lesion diagnosed by biopsy.
On the day of treatment by LU electrical excision procedure, T cells were harvested from the patient. The Ellis spot assay results of her CD eight T cell line indicate that the HPV 16, E 6 46 to 70 region contained a T cell epitope based on interferon gamma secretion levels 1.2 times 10 to the fifth. Epitope specific T cells were selected after limiting dilution on one fifth of the selected cells.
262 T-cell clones were harvested. The remaining selected cells were not used since the first attempt at T-cell cloning was successful. The Hiro put screening Ellis spot assay performed on 94 of these clones identified 62 screen positive clones.
The remaining T-cell clones were not tested since the first Ellis Spott assay identified peptide specific T-cell clones. The epitope specific nature of eight. Well growing screen positive clones were confirmed with the three 15 MER peptides covering the region individually.
All of the T-cell clones indicated positive with the HPV 16, E 6 51 to 65 peptides. Six of eight clones were positive with the E 6 46 through 60 peptide and none of the clones were positive. With the E 6 56 to 70 peptide shown here are Ellis spot assays with autologous LCLs infected with a recombinant vaccinia virus expressing HPV 16 E six protein or E seven protein.
These results demonstrate T cell clones recognize an endogenously processed E six epitope, a series of overlapping nine MER peptides covering the H PV 1646 to 65 region facilitated characterization for the minimal and optimal sequence of the T cell epitope. The best response was obtained with the E 6 53 to 61 peptide overlapping peptide epitopes identified the E 6 52 to 61 10 MER peptide as the minimal and optimal sequence most consistently. Interestingly, the E 6 53 to 61 peptide had equally strong response with the T-cell clone.
Number 79 dose response curves indicate the E 6 52 to 61 peptide retained positivity much more effectively than the E 6 53 to 61 peptide at lower peptide concentrations. In this example, subject A who was found to detect minimal and optimal sequence of E 6 75 to 83 was analyzed for restriction HLA class one molecule of the CD eight T cell epitope High positivity is seen with an LCL expressing the B 62 and CW three molecules. While background positivity is shown with an LCL expressing CW three, some regions of the HPV genome are well conserved.
For example, homologous sequences to the HPV 16, E 6 52 to 61 regions identified in subject one were present in 13 other high risk HPV types. Consistent with this, strong cross recognition was demonstrated for eight of 13 high risk HPV types once mastered, this technique can be done in seven weeks from the day of the blood draw if performed properly. Remember to use the sterile technique throughout the procedure.
Don't forget that working effects any virus can be hazardous and precautions such as wearing protective clothing should always be taken while performing the procedure. After watching this video, you should have a good understanding of how to characterize novel T-cell Es.
