Stem Cell-Derived Viral Ag-Specific T Lymphocytes Suppress HBV Replication in Mice

This method can help answer key questions in adoptive cell transfer of iPSC-derived viral antigen-specific T cell for effective suppression of HBV replication in mice. The main advantage of this technique is that the iPSC-derived viral antigen-specific T cells have a single type T cell receptor and naive phenotype. For differentiation of HBV-specific iPSC CD8-positive T cells, plate s183 T cell receptor gene-transduced iPSCs on an OP9-DL1/DL4 monolayer in alpha-minimum medium supplemented with 20%fetal bovine serum and murine Flt3 ligand for their co-culture in a cell culture incubator.

Monitor the co-culture regularly to assess the cell morphology. On day 28, remove the supernatant and floating cells, and detach the adherent cells with 0.25%trypsin. When the cells have lifted from the culture container bottom, stop the enzymatic reaction with eight milliliters of iPSC medium, and collect the cells by centrifugation.

Resuspend the pellet in 10 milliliters of fresh medium. Dilute the cells to a one iPSC-derived CD8-positive T cell to four s183 peptide-pulsed splenocyte ratio for a 40-hour incubation in the cell culture incubator. During the last seven hours, add four microliters of diluted brefeldin A to the culture, to block the transport processes during cell activation.

Stain the cells for flow cytometric analysis of the intracellular cytokines of interest according to standard protocols. For hydrodynamic HBV plasmid delivery through the tail vein, dilute 10 micrograms of HBV plasmid in an 8%equivalent of the body mass of PBS. Load the plasmid into one three-milliliter syringe equipped with a 26-gauge, 1/2-inch needle per mouse.

Heat HHD mice under a heat lamp for five minutes to dilate the tail veins, and carefully pull a mouse into a plastic restrainer. Locate a dilated lateral tail vein in the middle third of the tail, and insert the needle parallel to the vein to deliver the entire volume of plasmid through the tail vein over a three-to five-second period. For viral antigen-specific iPSC-derived CD8-positive T cell adoptive cell transfer, collect the iPSC CD8-positive T cells on day 22 of culture as demonstrated, and seed the cells onto a new 10-centimeter cell culture dish.

After 30 minutes in the cell culture incubator, filter the floating cells through a 70-micrometer nylon strainer, and count the viable cells. Dilute the cells to a 1.5 times 10 to the seven cells per milliliter of PBS concentration. Inject 200 microliters of cells into individual four-to six-week-old HHD mice, into the tail vein as just demonstrated.

On day 14 after cell transfer, perform a hydrodynamic HBV plasmid delivery as demonstrated. At the appropriate experimental endpoints post-infection, harvest the liver from each infected animal, and cut the livers into 0.5-by-0.5-by-0.3-centimeter tissue samples. Place the samples into 10%neutral buffer formalin for four to 24 hours, followed by decalcification in 2.5-molar formic acid for six to 24 hours.

Next, rinse the samples in xylene for three minutes, followed by two two-minute rinses in 100%ethanol, 95%ethanol, and deionized water. After the last water wash, decalcify the tissues in one-millimolar EDTA at 90 degrees Celsius, followed by a 30-minute incubation at room temperature. When the tissue has cooled, rinse the samples in PBS for four minutes, and use xylene and ethanol to deparaffinize and rehydrate the samples.

For immunofluorescent labeling, stain the sections with 200 microliters of HBV surface antigen-specific antibody for two hours at room temperature in a 75 to 100%humidified chamber. At the end of the incubation, wash the samples with five five-minute washes in fresh PBS for each wash. Label the cells with DAPI according to standard protocols before mounting the slides with coverslips for imaging by fluorescence microscopy.

For immunohistochemical analysis, stain the sections with hematoxylin and eosin according to standard protocols, and evaluate the infiltration of inflammatory cells into the liver by light microscopy. Flow cytometric analysis of the iPSC-derived CD3-positive CD8-positive population reveals that HBV T cell receptor transduction dramatically increases the generation of viral s183-specific CD8-positive T cells. After stimulation with T-depleted splenocytes pulsed with s183 peptide, CD4-negative CD8 single-positive iPSC-derived T cells produce large amounts of interferon gamma, as detected by intracellular flow cytometric analysis and ELISA.

After HBV infection by hydrodynamic injection, the DNA replication peaks on day six and reduces gradually, as observed by real-time PCR analysis. Further, viral replication is significantly reduced at all time points in mice that receive HBV viral antigenic-specific T cells compared to mice that receive control cells. HBV surface protein is also substantially decreased in mice that receive HBV viral antigen-specific pre-iPSC cytotoxic lymphocytes compared to mice treated with control cells, clearly demonstrating that viral antigen-specific iPSC CD8-positive T cells have the ability to reduce HBV replication in a murine model.

You should now have a good understanding of how to generate viral antigen-specific T cells from iPSC for adoptive immunotherapy.