In a Chronic HBV infection, CD4 T cells play important roles in both viral clearance and the returners. This method enables us to evaluate HBV specific CD4 T cell responses and identify HBV specific CD4 T cell epitopes, simultaneously. Demonstrating the procedure will be Jianmei Xiao, a research assistant.
And Xing Wan, a technician from Bilao. To begin, isolate peripheral blood mononuclear cells, or PBMCs, from blood, using Ficoll density gradient centrifugation at 800 times G for 20 minutes. Then, collect the granulocytes between the and red blood cell layer, using a pasteur pipette.
Transfer the thawed cell suspension to a 15 milliliter centrifuge tube, Add one milliliter of the pre warmed Benzonase RPMI 1640, drop wise, then slowly add another six milliliters. Rinse the cryo vial with two milliliters of Benzonase RPMI 1640 to retrieve the remaining cells. Then centrifuge the tube at 400 times G for 10 minutes.
Remove the supernatant and loosen the pellet by tapping the tube. Gently re-suspend the pellet in one milliliter of warm Bensenase NACE RPMI 1640. Mix the cells gently, and filter them through a 70 micrometer cell strainer, if any cell clumps are visible.
Count viable cells using trypan blue and a hemocytometer. Re-suspend the PBMCs and complete culture medium, containing 10 units per milliliter IL2, and 10 nanograms per milliliter IL7. Adjust the cell density to 1.5x10 to the 6 cells per milliliter.
Then plate the cells in 96-Well flat bottom plates at a density of 3x10 to the fifth cells per well. Add hepatitis B virus or HBV derived peptide pools to each Well. For the background and positive control Wells, add the same amount of solvent.
Incubate the plates at 37 degrees Celsius and 5%carbon dioxide. On day three, supplement the culture medium with 50 units per milliliter of IL2, and 10 nanograms per milliliter of IL7. On day seven, replace half of the culture medium with fresh medium containing:four micrograms per milliliter peptides, 100 units per milliliter IL2, and 20 nanograms per milliliter IL7.
On day 10, gently pipette the cells in each Well seven to nine times to dis-aggregate cell clusters. Count the number Of viable cells and transfer 2x10 to the fifth cells into each Well of a 96-Well round bottom plate for HPV specific CD4 T cell response analysis. For the residual cells in the 96-Well flat bottom plate, adjust the volume of the culture medium to 100 microliters by discarding excessive medium.
Then, supplement the culture with 100 microliters of fresh, complete culture medium, containing:four micrograms per milliliter peptides, 100 units per milliliter IL2, ans 20 nanograms per milliliter IL7. Continue culturing the cells at 37 degrees Celsius and 5%carbon dioxide for epitope identification on day 12. Wash the cells in the 96-Well round bottom plate by adding 200 microliters of RPMI 1640, centrifuging the plate at 550 times G for three minutes, and discarding the supernatant.
Repeat the wash twice using complete culture medium for the last wash. For each well of cells stimulated with a specific peptide pool, add 200 microliters of complete culture medium supplemented with the same peptide pool. For the Background Control Well, add complete culture medium, supplemented with one microliter per milliliter of DMSO.
For the Positive Control Well, add complete culture medium supplemented with one microliter per milliliter DMSO, 150 nanograms per milliliter PMA, and one micromole per liter of ionomycin. Incubate the plate at 37 degrees Celsius in 5%carbon dioxide for six hours. After one hour of incubation, add 1.37 micrograms per milliliter monensin to the culture.
After the six hour incubation is complete, centrifuge the plate at 550 times G for three minutes. Remove the supernatant and wash the cells once with 200 microliters of DPPs, as previously demonstrated. Stain for surface markers CD3, CD4 and CD8, and a viability marker.
After suspending the cells with vibrator, then refrigerate the plate at four degrees Celsius for 30 minutes. After washing the plate once with 200 microliters of DPBS, fix and permeabilize the cells, stain for intracellular cytokines, TNF Alpha and Interferon-gamma, and refrigerate the plate at four degrees Celsius for 45 minutes. Wash the cells once again, and re-suspend them in 150 microliters of flow cytometry buffer.
Then, acquire the flow cytometry data using a flow cytometer. Maintain allergenic B-lymphoblastoid cell lines, or BLCLs, in a T-75 flask. On day 12 of the PBMC expansion, count the number of viable BLCLs, and transfer them to 15 milliliter centrifuge tubes.
Centrifuge the cells at 350 times G for 10 minutes and remove the supernatant. Re-suspend the cell pellet and complete culture medium. Then aliquot the BLCLs to a 96-Well round bottom plate at 4x10 to the fourth cells per well, and 80 microliters of complete culture medium.
Add 10 micrograms per milliliter of a single peptide, and set two background controls. Peptide pulsing with HLADR blocking, and DMSO pulsing. Incubate the plates at 37 degrees Celsius and 5%carbon dioxide, for two hours.
Add 100 micrograms per milliliter Mitomycin C and incubate the plate at 37 degrees Celsius and 5%carbon dioxide for one hour. Wash the plate three times with 200 microliters of RPMI 1640 to remove unpulsed peptide and Mitomycin C, then re-suspend the cells in 120 microliters of complete culture medium, using vibrator. On day 12 of the PBMC expansion, transfer the PBMCs to a 96-Well round bottom plate.
Centrifuge the cells in the plate, remove the supernatant, and wash them twice with 200 microliters of RPMI 1640, as previously demonstrated. Re-suspend the PBMCs in each well with 210 microliters of complete culture medium. For the PBMC Wells chosen for epitope identification, aliquot 70 microliters of the cell suspension and mix with peptide pulsed BLCLs in three Wells, including two background controls.
Incubate the plate at 37 degrees Celsius and 5%carbon dioxide for six hours. After one hour of incubation, add 1.37 micrograms per milliliter monensin to the culture and bring the final volume in each well to 200 microliters with complete culture medium. In this representative example, the TNF Alpha and Interferon-gamma secreting CD4 T cell responses for peptide pool Core11 are lower than two times the background values, hence, considered negative.
Meanwhile, the responses for peptide pool Core09 are higher than two times the background, and considered positive. The gray background indicates Wells with a positive CD4 T cell response and candidate peptides for epitope identification are indicated in red, Core01 has the highest response rate, judging from both TNF Alpha and Interferon-gamma secreting CD4T cells in column peptide pools. Peptides C1 to 15, C31 to 45, C61 to 75, and C91 to 105 in this peptide pool, are set as candidate peptides as the row of peptide pools containing those peptides also show positive results.
PBMCs expanded with peptide pools Core07, 08, 09 and 10, are used for epitope identification of these peptides. Similarly, Core09 has the highest response rate in row peptide pools. All the peptides in this pool are set as candidate peptides and PBMCs expanded with peptide pools:Core01, 02, 03, 04, 05 and 06 are used for epitope identification of these peptides.
For peptide pool Core08 expanded PBMCs, after stimulation with peptide C31 to 45 pulsed BLCLs, the frequencies of TNF Alpha and Interferon-gamma secreting CD4 T cells are two times higher than background controls. Thus, peptide C31 to 45 is a verified HLADR restricted CD4 T cell epitope. It was there, additional expanded PBMCs remaining after epitope identification.
Fine marking of the identified epitopes can be performed using a panel of shortened peptides.
