During Zika virus infection, T cells can infiltrate immunoprivileged organs for virus elimination but may also contribute to immunopathogenesis, such as Guillain-Barre syndrome or testes injury. The main advantage of this technique is that it facilitates the tetramer-based detection of antigen-specific T cells in immunoprivileged organs, both during Zika virus infection and after vaccine immunization. In this model, Zika virus infection is initiated in interferon alpha/beta receptor knockout mice, allowing the tracking of Zika virus-specific T cells in the brain, testes, and spleen.
Using a tetramer staining approach, it is possible to investigate just characteristics of immunoprivileged organ infiltrating Zika virus-specific T cells to explore T-cell contributions to immunoprotection and immunopathogenic cells. For Zika virus infection, deliver one times 10 to the four focus-forming units of Zika virus in 100 microliters of PBS via retro-orbital inoculation into six-to eight-week-old interferon alpha/beta receptor knockout mice. Then, monitor the weight and clinical signs of the infected animals daily for 15 days.
Seven days post-inoculation, secure the infected animal in the supine position on a surgical foam, and use a sterile scalpel to make a skin incision along the midline of the abdomen. Use scissors to open the abdominal muscles, and gently extract the liver. Then, remove the spleen, and rinse the tissue three times in PBS to remove the blood.
After the last wash, place the spleen on 1.5 milliliters of ice-cold RPMI medium on ice until all of the spleens have been collected. To generate a single-cell suspension, place the spleens into a sterile, 40-micrometer mesh cell strainer on top of a 50-milliliter tube, and add two milliliters of ice-cold RPMI medium supplemented with 10%fetal bovine serum, or FBS, to the strainer. Then, use the plunger of a five-milliliter syringe to macerate the tissue through the filter, using fresh medium to flush the released cells through the mesh.
Transfer the single-cell suspension to a 15-milliliter conical tube for centrifugation, and resuspend the pellet in five milliliters of red blood cell lysis buffer. After five to six minutes at room temperature, stop the lysis with 10 milliliters of ice-cold RPMI plus FBS for a second centrifugation, and resuspend the pellet in 10 milliliters of complete medium for counting. Seven days post-inoculation, immobilize a euthanized, infected, male mouse in the prone position on a cutting board, and secure the scalp with straight one-by-two-teeth forceps.
Use iris scissors to make a midline incision on the scalp, exposing the skull, and use sharp tweezers to clamp the two sides of the orbits with sharp tweezers, fixing the brain. Placing one tip of sharp iris scissors into the foramen magnum, cut laterally into the skull on each side. Carefully cut from the same cavity up the midline, toward the nose, keeping the scissor tips as superficial as possible to avoid injuring the brain.
Lift the brain with forceps, and use sharp iris scissors to carefully dissect the cranial nerve fibers. Then, use the forceps to transfer the brain into a 15-milliliter tube containing five milliliters of ice-cold RPMI plus FBS. To isolate the testes, lift the abdominal skin with forceps, and use the iris scissors to make a longitudinal incision through the integument and abdominal wall and expose the lowermost part of the abdomen.
Push the testes up to the incision, and use tweezers to gently pull the fat layer, to expose a globular testis on both sides. Use the iris scissors to carefully dissect the fat layer and epididymis, and transfer the testes to a 15-milliliter tube containing five milliliters of ice-cold RPMI plus FBS. To generate a single-cell suspension from either of the harvested tissues, place the organ on a sterile cell strainer with a 100-micron mesh on top of a 50-milliliter tube, and add two milliliters of ice-cold RPMI plus FBS to the strainer.
Using the plunger from a five-milliliter syringe, mash the tissue, and wash the filter with fresh medium until the organ has been fully ground through the mesh. Transfer the single-cell suspension to a 15-milliliter tube for centrifugation, and resuspend the pellet in five milliliters of 30%density gradient medium. Carefully layer the cell solution over two milliliters of 70%density gradient medium in a new 15-milliliter tube, and separate the cells by density gradient centrifugation.
Then, transfer the cells from the interphase between the two density gradients to a new 15-milliliter tube containing 10 milliliters of cold RPMI plus FBS for another centrifugation, and resuspend the pellet in 10 milliliters of ice-cold RPMI/FBS for counting. To analyze the cells by flow cytometry, dilute the cells to a five times 10 to the six cells per 100 microliters in FACS buffer concentration, and incubate the cell samples with 10 microliters of anti-mouse CD16/CD32 blocking antibody per sample. After 10 minutes at room temperature, add 20 microliters of cells to the appropriate number of wells of a 96-well, round-bottom plate according to the experimental flow cytometry staining protocol, and add 20 microliters of the E-protein tetramet mix to each well for a 30-minute incubation at room temperature in the dark.
At the end of the incubation, add the cell surface antibodies of interest to the cells for a 30-minute incubation at four degrees Celsius, protected from light. Next, wash the cells two times with 200 microliters of FACS buffer per wash, and carefully resuspend the cells in each well with 200 microliters of fresh FACS buffer. Then, store the samples at four degrees Celsius, protected from light, until their analysis on a flow cytometer.
Pathological symptoms and signs such as myeloparalysis and motor paraparesis are observed in interferon alpha/beta receptor 1 knockout mice seven days post-inoculation with the Zika virus. Weight changes in the infected interferon alpha/beta receptor 1 knockout animals were monitored for 15 days, with weight loss first observed four days after infection and weight recovery beginning on day seven post-infection. Representative images of infected murine brains reveal obvious edema and hyperemia seven days post-inoculation with the Zika virus.
Representative images of murine Zika-infected testes demonstrate a gradual shrinking from day seven to 30 days post-infection. Histopathological analysis of Zika-infected brain and testicular tissue sections also illustrates the effect of the destructive pathological changes compared to uninfected controls. As expected from the macro and histopathological analyses, high viral loads are also detected in the brain and testes of Zika virus-infected mice by immunostaining.
That is accompanied by a robust CD3-positive T-cell infiltration into the brain post-Zika infection. Zika virus-specific CD3-positive, CD8-positive T lymphocytes are detected both in Zika virus-infected and vaccine-immunized mice by E-protein tetramer staining. E-protein tetramer-specific CD8-positive T cells can also be detected in the brain and testes seven days post-inoculation with Zika virus.
After its development, this technique paved the way for researchers in the study of pathogen-specific T-cell characterization in immunoprevalent organs during natural infections in animal models. This method can also be applied to other immune-prevalent organs, such as placenta or eye, as well as to both viral infection and cancer. Following this procedure, MHC-I tetramers can be used for the immunohistochemical or immunofluorescent detection of pathogen-specific T cells to access the distribution of brain-or testes-infiltrating T cells.
