Given the high mortality rate ( 50%) and potential danger of intrapersonal transmission, highly pathogenic avian influenza (HPAI) H5N1 epidemics still pose a significant threat to humans. findings provide a further understanding the mechanism underlying T cell-mediated innate and adoptive immune responses against HPAI H5N1 viral contamination, which helps to develop novel therapeutic strategies for the treatment of H5N1 infection in the future. mice provided by professor Mingzhao Zhu (Key Laboratory of Contamination and Immunity, Institute of Biophysics, Chinese Academy of Sciences). All mice were housed in an SPF facility. Lung injury was induced via the intratracheal instillation of AF vehicle or Limonin kinase inhibitor computer virus as previously reported (21). Briefly, WT and TCR-?/? mice were anesthetized by sodium pentobarbital and inoculated intranasally with 0.8 105 TCID50 H5N1 virus. At 4 days post contamination (DPI), mice were killed, and the lungs of each group of three mice were fixed in formalin and were then embedded in paraffin. Sections of 6 m thickness were obtained and stained with hematoxylin-eosin. At 4 DPI, the wet weight of the lungs of three mice was measured. The lungs were then heated to 68C for 24 h, and the dry weight of EDA the lungs was recorded; the wet/dry ratios were then calculated. The survival percentages and body weights in each group of 10 mice were monitored daily for 14 days. Survival data were analyzed by Kaplan-Meier survival analysis using GraphPad Prism 5 software. Expression of Recombinant HA (rHA) Proteins Monomeric and trimeric rHA proteins were expressed and purified using a baculovirus-insect cell system (Invitrogen, Thermo Fisher scientific, USA) as described previously (22). First, the HA ectodomain DNA fragment of A/Anhui/1/2005 (H5N1, Accession No. “type”:”entrez-nucleotide”,”attrs”:”text”:”DQ371928″,”term_id”:”87137936″,”term_text”:”DQ371928″DQ371928) and His tag were cloned into the transfer vector PacGP67b (BD Biosciences Pharmingen, USA) to allow the efficient secretion of monomeric rHA proteins. A new construct made up of the bacteriophage T4 fibritin Limonin kinase inhibitor fold on trimerization sequence was generated to allow the efficient secretion of trimeric rHA proteins as previously reported (23). Next, Sf9 cells were cotransfected with the monomeric or trimeric rHA transfer vectors and linearized baculovirus DNA (Invitrogen, Thermo Fisher scientific, USA) to produce recombinant baculoviruses made up of the HA genes. Transfection and computer virus amplification were carried out according to the baculovirus expression system manual. The supernatant from infected Sf9 cells Limonin kinase inhibitor was collected and purified by Ni-NTA chromatography (GE Healthcare, USA) against the C-terminal His tag. Western blotting was performed using anti-His or anti-HA antibodies to confirm the rHA proteins. To demonstrate that this expressed HA fragments were properly folded, they were analyzed by a Viscotek 270 Max GPC/SEC system according to the manufacturer’s instructions (Malvern, UK). Gel filtration chromatography was conducted using P4000 and P2500 columns Limonin kinase inhibitor (Malvern, UK) with a running buffer (pH 8.0) composed of 135 mm NaCl, 135 mm KCl, 1.5 mm KH2PO4, and 1.0 mm Na2HPO412H2O. Hemagglutination Assay Human erythrocytes were separated from whole blood of healthy donors. After isolation and washing, 50 l of a 0.75% human red blood cell (RBC) suspension was added to 50 l volumes of 2-fold serial dilutions of purified rHA proteins in a U-bottom 96-well plate (BD Falcon, USA; total volume, 100 l). Agglutination was read after incubation for 60 min at room temperature. As a control, phosphate-buffered saline (PBS) was used instead of rHA. Flow Cytometric Analysis Freshly isolated T cells were resuspended in PBS made up of 1% bovine serum albumin. The cells were then incubated with PE-conjugated anti-CD69 (BioLegend, USA) or isotype control antibodies for 20 min at 4C. After being washed with PBS for three times, the cells were analyzed on an Accuri C6 flow cytometer (BD Biosciences, USA). The data are presented as either the percent positive cells or the mean fluorescence intensity. IFN- Secretion Assay A total of 1 1 106 T cells per well were seeded into 48-well plates Limonin kinase inhibitor and were then treated with PBS, purified rHA proteins for 12, 24, and 48 h. Cell-free supernatants were collected,.