Sample collection
A total of 74 samples from 64 individuals were enrolled in this study with ages ranging from 14 to 74 (Table 1). Another 52 healthy volunteers were collected from the physical examination population. The patients’ samples were recruited at the Xi’an eighth hospital from November 2012-Januarary 2024. Clinical diagnosis of HFRS was confirmed by the detection of HTNV-specific IgM or IgG antibodies. Peripheral blood samples and plasma were collected as previously described55. The ethics committees for human experimentation approved all experiments and sample collection that involved human participants in our study. Four clinical types are classified including mild, moderate, severe, and critical according to the diagnostic criteria from the Prevention and Treatment Strategy of HFRS. The viral load was measured using established protocols56.
All the sample collection procedures were in accordance with the ethical standards of the responsible committee on human experimentation (Xijing Hospital, First Affiliated Hospital of Fourth Military Medical University, Xi’an, China (NO.KY20243512-1, NO.KY20224214, and NO.KY20224212). Informed consent was obtained from the patients or guardians of patients. All the data were analyzed anonymously. All ethical regulations relevant to human research participants were followed.
Mice
The MAIT cells deficiency mice (C57BL/6JCya-Mr1em1/Cya mice, MR1-/- mice) were purchased from Cyagen Biosciences (Suzhou) created by CRISPR-assisted gene targeting strategies. Age- and sex- matched littermates were used in the study. Mice were housed in cages with microisolator tops on ventilated or static racks in a specific pathogen-free facility. Ambient temperature was 20 °C, on a 12-h light dark cycle with automatic light control. Animals were randomized for treatments that were blinded to personnel carrying out the injections and physiological function analysis. Appropriate animal sample size was determined using sample size calculator (http://www.lasec.cuhk.edu.hk/sample-size-calculation.html). All animal works were approved by the Institutional Animal Ethics and Use Committee of the Fourth Military Medical University (NO. 20230275). We have complied with all relevant ethical regulations for animal use.
Cell culture
MAIT cells were enriched by using PE-Vα7.2 antibody and anti-PE Nanobeads (Biolegend) from the peripheral blood mononuclear cells (PBMC) isolated from peripheral blood of human. The MAIT cells were cultured in RPMI 1640 conditioned medium containing 10% fetal bovine serum (Hangzhou Sijiqing Company), 1% Penicillin–streptomycin (Corning), 10 U/ml IL-2 and 20 U/ml IL-7. For stimulation, 20 μM 4μ8c (the inhibitor of IRE1α pathway) and 500 nM thapsigargin (the activator of ER stress) were used for 24 h. For activation, 50 ng/ml IL-12 and 50 ng/ml IL-18 were stimulated for 24 h as reported before.
Human umbilical vessel endothelia cells (HUVECs) were collected as previously described and stored in our lab57. The HTNV infected-HUVECs were co-cultured with the serially diluted supernatants of activated MAIT cells. Three days after infection, the protein level of the nucleocapsid protein (NP) of HTNV in HUVECs were measured by Western blot.
HTNV infection
For in vitro infection, Hantaan virus 76-118 strain was propagated in Vero E6 cells using standard methods and the titer was determined using plaque assays58. The mock HTNV was prepared by irradiating the HTNV with 1.1×104 Gy with 60Co as previously reported. 1 ~ 2×105 MAIT cells or PBMCs were seeded in 24 well plate and were exposed to HTNV at multiplicity of infection (MOI) of 2 for 2 h, cultured at 37 °C at 5% CO2 for 24 h. The cells in control group were left unstimulated. Next, cells were washed and then cultured as above described.
For in vivo infection, male mice aged 5 weeks weighed 20 ± 2 g were chosen in each group. They were challenged with intramuscular injection of the HTNV 76-118 strain (1 × 105 pfu/mouse). Seven days post infection, the mice were sacrificed. Tissue samples from six major organs, including cerebrum, heart, liver, spleen, lung and kidney, were harvested for the HTNV NP detection. The organ tissue lesions were also observed using HE staining.
RNA isolation and qPCR
2×105 MAIT cells were seeded in 24 well plate and infected with HTNV for 24 h. The RNA was extracted using TRIzol reagent (Invitrogen) and following its protocols. qPCR was performed using the SYBR Green QPCR Master Mix (Takara) on the Roche LightCycler 480 platform. The xBP1s primer sequences were as follows, Forward, 5’-GCAGGTGCAGGCCCAGTTGT-3’, Reverse, 5’-TGGGTCCAAGTTGTCCAGAATGC-3’. The results were normalized to the β-actin mRNA level.
Western blot
The collected HUVECs were lysed in RIPA buffer. Western blot (WB) was performed using conventional procedures with 1A8 antibody specific to NP of HTNV, which was kindly provided by the Department of Microbiology, Fourth military Medical University58. After developing with 1A8 antibody, the membrane was washed, stripped and re-probed for GAPDH. Band intensity was quantified by ImageJ software. The NP level, pro-caspase1 and cleaved-caspase1 were normalized to the corresponding GAPDH level. Uncropped images can be found at the Supplementary Fig 6.
The sorted MAIT cells from HFRS patients or healthy donors were collected and the similar procedures were performed with the Caspase-1 primary antibody.
Single-cell RNA sequencing (scRNA-seq) data processing and analysis
Sequencing was done as previously described55. The data can be found at Public Gene Expression Omnibus database (GSE161354). The information of samples collection was concluded before55. The MAIT cells cluster was defined and analyzed as previously described12.
Flow cytometry analysis
For cell surface staining, 1×106 PBMCs from HFRS patients and health donors (setting as normal controls) were stained as previously described. Briefly, antibodies cocktails were added to the cell suspension in 100 μl flow cytometry staining buffer. The primary antibodies used in the study were summarized in the Supplementary Table 1. After incubating the cells at 4 °C in the dark for 30 min, the cells were washed once with staining buffer at 4 °C. Next, cells were subjected to flow cytometry in 100 ul staining buffer.
For intracellular staining, 2×106 PBMCs from HFRS patients and health donors (setting as normal controls) were stained with surface markers at first place. Next, cells were fixed and permeabilized using an intracellular staining kit (eBioscience). The cells were then incubated with Cleaved Caspase 3 (Cell Signaling Technology), NLRP3 (Cell Signaling Technology) or TMCO1 (Invitrogen) antibodies in permeabilization buffer at 4 °C for 30 min. After incubating with corresponding second antibodies, cells were resuspended in 100 ul staining buffer.
For apoptosis assay, 1×106 PBMCs were stained with MAIT surface markers, then Annexin V and 7-AAD staining were performed following the instructions.
For FLICA-caspase 1 staining assay, 1×106 PBMCs were incubated with FLICA-Caspase1 (ImmunoChemistry Technologies) for 1 h at 37 °C in RPMI 1640 medium. After washing twice with 1×washing buffer supplied in the kit, the cells were then stained with MAIT cells’ surface markers as mentioned above.
For measurement of ER Ca2+ stored in MAIT cells, 3×106 PBMCs were prewashed with RPMI 1640 medium containing 5 mM EGTA. These calcium-free medium was used for the whole experiment. Then the cells were loaded with 2 μM Fluo3 and Fura Red dye, CD3-PE-Cy7, CD8-APC-Cy7, Vα7.2-PE, and CD-161-efluro450 in the above medium for 30 min at 37 °C. After washing with the above medium, the cells were resuspended in 200 μl RPMI 1640 medium containing 5 mM EGTA. The samples were placed on the cytometer, recorded 1 min to get the baseline intensities. Then we quickly added 3 μM of ionomycin to the tube. Samples were mixed quickly and recorded for another 4 min.
The samples were acquired with ACEA NovoExpress cytometer (Agilent Bio). The obtained data was analyzed using the FlowJo software (TreeStar), with results expressed in term of a percentage value of the positive cells or mean fluorescence intensity (MFI). In some assay, the images were taken on an Amnis ImageStream X mark II imaging flow cytometer.
Immunofluorescence staining assay
For cells, 2×105 sorted MAIT cells under different conditions were spined onto poly-lysine slides by Cytospin (Xiangyi Centrifuge Instrument Co., Ltd., China). Then the cells were fixed with 4% paraformaldehyde for 5 min at room temperature (RT). After washing slides with PBS for twice, the cells were permeabilized with 0.5% triton x-100 in PBS for 20 min at RT. After rinsing twice with PBS, the slides were blocked by goat serum (1:20) for 20 min at RT. The slides were incubated with primary antibodies (Supplementary Table 1), which were 1:200 diluted in PBS containing with 2%BSA + 0.1% Triton X-100 (antibody dilution buffer). After incubation the slides at 4 °C overnight, the slides were rinsed with PBS for five times and were incubated with corresponding fluorescent secondary antibodies.
For tissues, immunofluorescent staining was carried out by Lilai biomedicine. 1A8 antibody or CD31 antibody (Lilai provided) was used as primary antibody. CY3 labeled goat anti-mouse antibody or FITC labeled goat anti-rabbit antibody was used as secondary antibodies. The images were collected by confocal microscopy (Olympus or Nikon, Japan).
The positive cells were counted when they colocalized with DAPI and localization proteins (CD31, HLA-I or KDEL).
HE staining assay
4% paraformaldehyde fixed tissue sections were stained with a hematoxyline and eosine (H&E) using standard techniques. The digital microphotographs were taken at a magnification of ×100 using a computer-assisted image analyzing system. The sections were scanned using automatic digital slice scanning system (Science, Shandong, China).
Enzyme-linked immunosorbent assay (ELISA)
The detection of IL-18 (NeoBioscience, EHC127) and IL-1β (NeoBioscience, EHC002b) in the supernatant were performed using ELISA kits. TECAN Infinite 200pro was used to determine the absorbance at 450 nm. The experiment was performed according to the manufacturer’s instructions.
Statistics and Reproducibility
All data analyzed were performed using the GraphPad Prism 8 software. For the correlation analysis, nonparametric Spearman’s correlation analysis was used. For the comparisons, two-tailed students t-test was used for comparison between the two groups. One-way analysis of variance (ANOVA) was used for comparison between three or more groups. Paired t test was used when using paired samples. Data are shown as the mean value±standard error of the mean (SEM) based on more than three independent experiments. In all tests, values of *p < 0.05, **p < 0.01, and ***p < 0.001 were considered statistically significant.
Reporting summary
Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.
