key: cord-0766714-km6fnf5u authors: Zhang, Yongli; Zhang, Hangjie; Ma, Wenqiang; Liu, Kefang; Zhao, Min; Zhao, Yingze; Lu, Xuancheng; Zhang, Fuping; Li, Xiangdong; Gao, George F.; Liu, William J. title: Evaluation of Zika Virus-specific T-cell Responses in Immunoprivileged Organs of Infected Ifnar1(-/-) Mice date: 2018-10-17 journal: J Vis Exp DOI: 10.3791/58110 sha: 4090c4a0e8cead64dd9e44a1610fb4f557fefdcf doc_id: 766714 cord_uid: km6fnf5u The Zika virus (ZIKV) can induce inflammation in immunoprivileged organs (e.g., the brain and testis), leading to the Guillain-Barré syndrome and damaging the testes. During an infection with the ZIKV, immune cells have been shown to infiltrate into the tissues. However, the cellular mechanisms that define the protection and/or immunopathogenesis of these immune cells during a ZIKV infection are still largely unknown. Herein, we describe methods to evaluate the virus-specific T-cell functionality in these immunoprivileged organs of ZIKV-infected mice. These methods include a) a ZIKV infection and vaccine inoculation in Ifnar1(-/-) mice; b) histopathology, immunofluorescence, and immunohistochemistry assays to detect the virus infection and inflammation in the brain, testes, and spleen; c) the preparation of a tetramer of ZIKV-derived T-cell epitopes; d) the detection of ZIKV-specific T cells in the monocytes isolated from the brain, testes, and spleen. Using these approaches, it is possible to detect the antigen-specific T cells that have infiltrated into the immunoprivileged organs and to evaluate the functions of these T cells during the infection: potential immune protection via virus clearance and/or immunopathogenesis to exacerbate the inflammation. These findings may also help to clarify the contribution of T cells induced by the immunization against ZIKV. The ZIKV is a mosquito-borne flavivirus that was first isolated in 1947 in Uganda from a febrile rhesus macaque. Recently, the ZIKV has become a public health emergency, due to its rapid dissemination in the Americas and its unexpected link to microcephaly and Guillain-Barré syndrome 1, 2, 3 . From epidemiological data, the ZIKV has been suspected to be the cause of the Guillain-Barré syndrome in around 1 per 4,000 infected adults 4 . Moreover, a correlation between the ZIKV and testes infection/damage in the mouse model has been demonstrated, suggesting that the ZIKV infection, under certain circumstances, can bypass the blood-testis barrier and eventually lead to male infertility 5 . These findings highlight the importance of completely understanding the induction of protective or pathologic immune responses during a ZIKV infection. Much remains to be learned about the cellular immune responses to the ZIKV. CD4 + and CD8 + T-cell responses to the capsid, envelope, and nonstructural protein 1 (NS1) have been observed in ZIKV-infected monkeys and humans 6, 7, 8 . In mice, several studies have indicated that CD8 + T cells play a protective role in controlling the ZIKV replication 9, 10, 11 . Importantly, Jurado et al. demonstrated that a ZIKV infection results in the breakdown of the blood-brain barrier and perivascular infiltration of CD8 + effector T cells within the testes of Ifnar1 -/mice. Furthermore, they showed that CD8 + T cells instigate ZIKV-associated paralysis and appear to play a role in the neonatal brain immunopathology. In a previous study, we prepared the ZIKV-E 294-302 tetramer and showed that ZIKV-specific CD8 + T cells exist in the brains and spinal cords of ZIKV-infected Ifnar1 -/mice, which may have important implications for the design and development of ZIKV vaccines 10 . In response to the urgent need for vaccination to prevent ZIKV infection, several vaccines are in the preclinical stages of development, including RNA vaccines, recombinant vector-based vaccines, and purified protein subunit vaccines. The plasmid DNA vaccine is in phase 1 clinical trials 12 . The evaluation of safety and efficacy of ZIKV vaccines is, therefore, important. One advantage of the vaccines is their ability to elicit specific T-cell responses, which may be important for protection against the ZIKV. By using ZIKV-derived T-cell epitope-related tetramers, the T-cell 5. Use sharp Iris scissors to carefully cut from the same cavity, up the midline, toward the nose. Try to keep the end of the scissors as superficial as possible to avoid injuring the brain. 6. Lift the brain with forceps and use sharp Iris scissors to carefully dissect the cranial nerve fibers. Remove the brain with forceps and place it in a 15 mL tube containing 5 mL of ice-cold RPMI/10% FBS medium. 7. Grab the abdominal skin with forceps and use sharp 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. Gently pull the fat layer with tweezers and expose a globular testis on both sides. 8. Use sharp Iris scissors to carefully dissect the fat layer and epididymis. Place the testes in a 15 mL tube containing 5 mL of ice-cold RPMI/10% FBS medium with forceps. 9. To generate a single-cell suspension from the brain or testes, place the organ on a sterile cell strainer with a 100 µm mesh on top of a 50 mL tube and add 2 mL of ice-cold RPMI/10% FBS medium. Using the plunger of a 5 mL syringe, mash the organ and add medium until the organ has been fully ground through the mesh. 10. Transfer the cell suspension to a 15 mL tube and centrifuge it at 600 x g for 5 min at 4 °C. Remove the supernatant. 11. Resuspend the cells with 5 mL of 30% density gradient medium and, then, add them very slowly to 2 mL of 70% density gradient medium in a 15 mL tube. 12. Switch off the brake and centrifuge the tubes at 4 °C at 800 x g for 30 min. Obtain the lymphocytes from the middle layer. 13. Transfer the interphase to a fresh 15-mL tube, add 10 mL of cold RPMI/10% FBS medium, and centrifuge the tube at 300 x g for 10 min at 4°C . Remove the supernatant. 14. Resuspend the cells with 10 mL of ice-cold RPMI/FBS medium and centrifuge them at 600 x g for 5 min at 4 °C. Remove the supernatant. 15 . Resuspend the cells with 10 mL of complete RPMI medium and count the number of cells as in step 3.9. mM NaCl] to the chamber and concentrate it to a final volume of 30-50 mL. 4. Transfer the refolding solution to a centrifuge tube and spin it at 2,500 x g for 15 min at 4 °C. 5. Carefully transfer the supernatant to a 10 kDa centrifugal filter and further concentrate it to a final volume of 500 µL at 2,500 x g for 30 min. 6. Transfer the supernatant to a fresh tube and spin it at 12,000 x g for 15 min. Purify the protein with S200 10/300 GL gel filtration chromatography. 7. Collect the MHC complex peak and concentrate it to a final volume of 350 µL. 8. Apply the multimerized reagents into a 100 kDa spin tube and concentrate it by centrifugation at 2,000 x g at 4 °C to a volume of <100 µL. 9. Dilute the sample in the spin tube to 4 mL using PBS (pH 8.0) and concentrate it again to <100 µL. 10 . Repeat the buffer exchange step in PBS (pH 8.0) 4x. 11. Fill up the total volume again to 500 µL using PBS (pH 8.0). Concentrate the sample to an estimated concentration of 2-2.5 mg/mL at 2,000 x g at 4 °C. Store the sample in the dark at 4 °C. Following these methods, we have developed a murine model for ZIKV infections. Ifnar1 -/mice at 6-8 weeks of age were infected with 1 x 10 4 focus-forming units (FFU) of the ZIKV by retroorbital injection. Pathological symptoms and signs (Figure 1A) , as well as weight changes ( Figure 1B) , were observed in the Ifnar1 -/mice after an infection with the ZIKV. The murine brains showed obvious edema and hyperemia ( Figure 1C) . Meanwhile, the testes shrank gradually ( Figure 1D) . Furthermore, pathological changes and the destruction of tissue were found in the brain and testes (Figure 2A) . We performed an immunofluorescence assay to detect the ZIKV in the brain and testes (Figure 2B) . High viral loads were detected in the brain and testis by immunostaining ( Figure 2B ). Immunohistochemistry showed a robust infiltration of CD3 + T cells into the mice brain after the infection with the ZIKV (Figure 2C) . To detect and evaluate ZIKV-specific T-cell responses, we prepared a mouse MHC-I H-2D b -E 294-302 tetramer. The peptide E 294-302 can help the H-2D b renature properly and yield a high amount of the soluble MHC-I (Figure 3A) . In the shift assay, a high efficiency in biotinylation could be observed ( Figure 3B) . Subsequently, three streptavidin fluorescence (APC, PE, and BV421)-tagged pMHC-I tetramers were produced to detect ZIKV-specific T cells (Figure 3C) . The PE-labeled tetramer had a higher efficacy to detect the specific CD8 + T cell compared to the APC-and BV421-labeled tetramers, though the difference was not statistically significant. Using the E 294-302 tetramer, we detected ZIKV-specific T lymphocytes in the spleen of the infected mice by flow cytometry at 7 d post-inoculation of the ZIKV (3.49 ± 0.45%). Also, similar to the method described in section 3 of this protocol, with 4 weeks post-immunization of AdC7-M/E vaccine, ZIKV-specific T lymphocytes were detected in the spleen (6.89 ± 1.36%) (Figure 4) . Furthermore, we detected the lymphocytes infiltrated into the immunoprivileged organs, such as the brain and testes, after the ZIKV infection. The gates were set to select for CD3 + CD8 + T cells in total lymphocytes of the brain and testes. A high ratio of the E 294-302 tetramer-specific T cells could be detected in the brain (42.2% in CD3 + CD8 + T cells) and the testicular (26.4% in CD3 + CD8 + T cells) lymphocytes ( Figure 5 ). Immunogenic T-cell epitope plays a significant role in cellular immunity against pathogens 23 . Thus, the detection of ZIKV-specific T cells in immunoprivileged organs is a critical methodology to understand T-cell responses against the natural ZIKV infection. Meanwhile, T-cell response detection is an excellent tool to investigate the efficacy of the viral vaccine. Here, we show a comprehensive protocol to visualize the experiments, which include the isolation of lymphocytes from the spleen, brain, and testes of ZIKV-infected mice, the preparation of the immunodominant epitope E 294-302 tetramer, and the recognition of ZIKV-specific CD8 + T cells in immunoprivileged organs of ZIKV-infected mice. A previous study showed that a live ZIKV or its RNA can be detected in the semen of male patients, which indicates that the ZIKV can bypass the blood-testis barrier and replicate itself in the reproductive system 24 . Previously, we also showed that the ZIKV can cause testes damage and lead to male infertility in mice 25 . ZIKV infection can lead to viremia in rhesus monkeys, and the viral RNA can be detected in the central nervous system (CNS), as well as in the visceral organs. Immunohistochemistry revealed that ZIKV-specific antigens were presented in the CNS and the multiple peripheral organs 26 . Also, in murine models, ZIKV infection can induce a robust antiviral CD8 + T-cell response in the spleen and CNS 26 . Compared to previous work, this study establishes systematic methods to detect ZIKV-specific CD8 + T-cell responses in the brain and testes, which are immunoprivileged sites. It is important to assess the functionality of virus-specific T cells in the immunoprivileged organs of the ZIKVinfected mice. The usage of tetramers to detect ZIKV-specific CD8 + T-cell responses in immunoprivileged organs would greatly enhance our understanding of ZIKV infections and their host immune responses. Using E 294-302 tetramer, virus-specific T cells in brain and testis can be isolated by flow cytometry, to investigate the cellular mechanisms of the protection and immunopathogenesis during a ZIKV infection. Meanwhile, it is helpful for researchers to investigate further the functions of the CD8 + T cells to control the ZIKV, or to enhance the immunopathogenesis in these organs during ZIKV infection. To analyze the antigen-specific murine CD8 + T-cell responses in the immunoprivileged organs, we prepared H-2D b -E 294-302 tetramer and detected the CD8 + T cells by flow cytometry. Tetramers are a powerful tool to detect antigen-specific T cells. Here, three types of fluorochromeconjugated streptavidin (APC, PE, and BV421) were generated. Although there are no statistically significant differences in the APC-, BV421-, and PE-labeled tetramers for detecting antigen-specific T cells, PE-labeled pMHC-I tetramers yielded the best results. Hence, the PE-labeled tetramer was used throughout this study. Interestingly, based on the PE-labeled H-2D specific T cells in both the brain and testes, which indicate the migration ability of the virus-specific T cells from the blood to immunoprivileged organs. However, there are some limitations to the protocol. The H-2D b -E 294-302 tetramer is not useful for human T-cell detection, because tetramer detection is dependent on MHC restriction. The screening of immunodominant HLA-restricted peptides is still needed. Besides, retro-orbital infection is effective for a ZIKV infection but might be not a convenient operation for some investigators. Thus, other routes of infection, including peritoneal, subcutaneous, or intravenous, are also recommended. In the protocol described here, a critical step is the isolation of monocytes from brain and testis. It is important to acquire high-quality lymphocytes; thus, it is important to pay attention to, for example, the centrifugal speed, the strength of the grinding tissue, and the dissection of the brain and testis tissue. Besides, for tetramer preparation, protease inhibitors (PMSF, pepstatin, leupeptin) are helpful when protecting a protein from being degraded. Therefore, it makes sense to add a protease inhibitor to the refolding buffer and exchange the buffer during the process of the tetramer preparation. In conclusion, we present the methods of detecting antigen-specific T-cell responses in the immunoprivileged organs of the Ifnar1 -/mouse model for a ZIKV infection. This platform can be widely applied to investigate the immune mechanisms of emerging and re-emerging viruses which can bypass the barriers between the blood and the immunoprivileged organs. Moreover, this study may pave the way for the future development of candidate vaccines and immunotherapies. The authors have nothing to declare. Collect the brain and testis tissues of the ZIKV-infected Ifnar1 -/-mice 7 d post-inoculation and fix them in 4% neutral-buffered formaldehyde. CAUTION:Paraformaldehyde is toxic Embed the tissue in paraffin Stain the tissue with hematoxylin and eosin (H&E) Immunohistochemistry assay 1. Deparaffinize, rehydrate, and antigen-retrieve the tissue sections Treat the tissue sections with 3% H 2 O 2 in PBS (pH 7.6) for 10 min and block them with 1% bovine serum albumin (BSA) for 10 min Incubate the tissue sections with rat anti-mouse CD3 antibody (dilution: 1/1,000) for 8 h at room temperature and, then Rinse the tissues with PBS and, then, incubate them with 3 drops of biotinylated secondary antibody (dilution: 1/1,000) for 2 h at room temperature, followed by 3 drops of avidin-biotin-peroxidase (dilution: 1/200) at room temperature for 30 min 3 drops of 30, 30-diaminobenzidine tetrahydrochloride (dilution: 1/1,000), as described previously 22 Immunofluorescence assay 1. Air-dry the frozen testis sections (6 mm) for 10 min at room temperature. 2. Fix them with ice-cold acetone for 10 min Wash the sections with PBS for 3x and block them with a blocking buffer (1% BSA, 0.3% Triton, 1x PBS) at 37 °C for 30 min Incubate the tissue sections with primary antibody (Z6) (20 µg/mL) at 4 °C overnight Rinse the tissues with PBS and apply the secondary antibody (dilution factor: 1/200) for 1 h at 37 °C Wash the tissue sections with PBS and counterstain them for nuclei using 4', 6-diamidino-2-phenylindole (DAPI) (dilution factor: 1/1,000), following the manufacturer's instructions Zika virus. I. 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