Mice
Gp96fl/fl mice were provided by Professor Zihai Li (Ohio State University, United States of America). Ncr1-iCre mice were purchased from Biocytogen (Beijing, China). Gp96fl/fl mice were crossed with Ncr1-iCre mice to generate NK-specific gp96-deficient mice (referred to as KO mice); age and sex-matched gp96fl/fl mice were used as control (referred to as WT mice). All mice used were 8 to 12 weeks old, had a B6 background, and were housed in the specific pathogen-free facility at the Institute of Microbiology, Chinese Academy of Sciences. All animal experiments were approved by the Institutional Animal Care and Use Committee (permit number PZIMCAS2011001).
Cell Lines And Plasmid Construction
B16F10 cells were cultured in-house. The HEK293 cell line, NK92 human NK cell lines, MC38 colon cancer cells, and LLC lung cancer cells were purchased from the Cell Resource Center, Peking Union Medical College. The cell line was checked free of mycoplasma contamination by PCR and culture. Its species’ origin was confirmed with PCR. The cell line’s identity was authenticated with STR profiling (FBI, CODIS). All the results can be viewed on the website (http://cellresource.cn). B16F10 cells were cultured in RPMI 1640 medium with 10% fetal bovine serum (FBS), penicillin, and streptomycin (100 IU/ml). HEK293, MC38, and LLC cells were cultured in Dulbecco’s modified Eagle medium with 10% FBS, penicillin, and streptomycin (100 IU/ml). NK92 cells were cultured in α-MEM with 12.5% FBS, 12.5% horse serum, penicillin, and streptomycin (100 IU/ml), and IL-2 (10 ng/ml). All plasmids used in this paper were purchased by iGene Biotechnology Co., Ltd.
Western Blot And Co-immunoprecipitation Assay
The stimulated NK92 cells or sorted splenic NK cells were harvested, and an equal number of cells were directly lysed. The following primary antibodies were used for Western blot: Antibodies to GAPDH (D16H11), β-actin (8H10D10), phospho-S6 (D57.2.2E), phospho-AKT (Ser473) (D9E), Phospho-Stat5 (Tyr694) (D47E7), gp96 (D6X2Q), Trim28 (C42G12), His-Tag (D3I1O) and LC3B (D11) were purchased from Cell Signaling Technology (Danvers, USA). Antibodies specific for Eomes was purchased from Santa Cruz Biotechnology (CA, USA). Antibody specific for c- Myc-tag (5D10), DDDDK-tag (1A8), GFP-tag (3A10) and HA-tag (4G3) was purchased from Bioworld (Nanjing, China).
For endogenous Co-IP, cells were lysed with 0.25% NP-40 lysis buffer (20 mM Tris-HCl, 125 mM NaCl, 5 mM MgCl2, 0.2 mM EDTA, 12% Glycerol, and 0.25% Nonidet P-40). Equal amounts of total protein were incubated with indicated antibodies or normal mouse IgG overnight at 4°C, and then 40 µl of protein A/G beads were added for an additional 2 h of incubation. For exogenous Co-IP, anti-HA beads (or anti-flag beads, or anti-myc beads) were added to equal amounts of total protein and incubated for 3 h. Beads were centrifuged (800 rpm for 2 min) and washed five times using wash buffer (20 mM Tris-HCl, 125 mM NaCl, 5 mM MgCl2, 0.2 mM EDTA, and 0.1% Nonidet P-40). The beads were heated at 100°C for 10 min before SDS-PAGE and immunoblotting.
In vivo ubiquitination assays
For the in vivo ubiquitination assay, HEK293 cells were co-transfected with expressing plasmids encoding Myc-Eomes and Trim28. Cells were grown overnight and treated with 20 nM Bafilomycin A1 (BafA1) for 6 h before harvesting. Cell lysates were immunoprecipitated using anti-Myc resin, followed by Western blot analyses with ubiquitin, ubiquitin-Lys 48-only or ubiquitin-Lys 63-only.
Flow Cytometry
Single-cell suspensions were stained with the appropriate monoclonal antibody in phosphate-buffered saline containing 5% serum. To detect phosphorylated signaling proteins, NK cells were fixed with Phosflow Lyse/Fix buffer, followed by permeabilization with Phosflow Perm buffer III (BD) and staining with antibodies. All other intracellular proteins were stained according to the manufacturer’s instructions using Foxp3/Transcription Factor Staining Buffer Set Kit (eBioscience). Fortessa and FACSAria III (BD Biosciences, San Diego, USA) were used for analysis and cell sorting, with dead cells excluded by the LIVE/DEAD Fixable Violet Dead Cell Stain Kit (Invitrogen, Carlsbad, USA). Antibodies specific for anti-mouse CD3 (17A2), anti-mouse CD49b (DX5), anti-mouse NK1.1 (PK136), anti-mouse/human CD11b (M1/70), anti-mouse CD45 (30-F11), anti-mouse CD3(17A2), anti-mouse CD27 (LG.3A10), anti-mouse KLRG1 (2F1/KLRG1), anti-mouse CD122 (TM-β1), anti-mouse NKp46 (29A1.4), anti-mouse IFN-γ (XMG1.2), anti-mouse CD19 (6D5), anti-CD4 (GK1.5), anti-Foxp3 (FJK-16s) and anti-mouse CD107a (LAMP-1) were purchased from BioLegend (San Diego, USA). Antibodies specific for anti-Eomes (Dan11mag), anti-Id2 (ILCID2), anti-T-bet (eBio4B10 (4B10)), anti-CD11c (N418), anti-F4/80 (BM8), anti-CD8a (53 − 6.7), anti-human EOMES (WD1928), anti-human CD3 (UCHT1), anti-human CD56 (CMSSB), anti-human CD27 (O323) and anti-human CD45 (HI30) were purchased from Thermo Fisher Scientific. Antibodies specific for phospho-S6 (N7-548) and phospho-AKT (Ser473) (M89-61) were purchased from BD Biosciences (San Diego, USA). PE-conjugated grp94 monoclonal antibody (9G10) was purchased from Enzo Life Sciences.
Rna-seq And Analysis
CD3−NK1.1+DX5+ NK cells were sorted to a typical purity of > 98%. RNA-seq and bioinformatics analysis were conducted by Shanghai Biotechnology Corporation.
Single-cell Rna Sequencing And Data Processing
Freshly isolated splenocytes were stained with anti-CD3 (17A2), and anti-NK1.1 (PK136) antibodies and NK cells were sorted to > 99% purity using a BD FACSAria™ III. Splenic NK cells pooled from three mice of each indicated genotype were enriched by FACS for library preparation, and scRNA-seq was performed by Shanghai Biotechnology Corporation. The final library pool was sequenced on the Illumina NovaSeq 6000 instrument using 150-base pair paired-end reads. Raw sequencing data were converted to FASTQ files and aligned to the mouse genome reference sequence (GRCH38). The 10X Genomics Cell Ranger (3.0.1 version) was used to demultiplex samples, process barcodes, and generate a digital gene-cell matrix from this data. The output was then imported into the Seurat (v3) R toolkit for quality control and downstream analysis. The percentage of ribosome genes is 0.326 and 0.438% in gp96fl/fl and Ncr1-iCre gp96fl/fl cells, respectively. Cells with less than 200 genes and cells with above 20% mitochondrial genes were removed as low-quality cells. Cells with abnormally high unique molecular identifiers or gene numbers were also identified as putative doublets and removed. After quality control and filtering steps, 7349 cells from Ncr1Cre mice and 8350 cells from Ncr1Cregp96fl/fl mice were used for further analyses. The Louvain algorithm was used for the unsupervised computational analysis of scRNA-seq data.
Generation Of Bm Chimeras
To generate mixed BM chimeras, BM cells were isolated from the femurs and tibias of CD45.1+WT and CD45.2+ Ncr1Cregp96fl/fl mice. WT recipient mice (CD45.1+CD45.2+) were sub-lethally irradiated (7 Gy) and intravenously transplanted with a mixture (1:1) of WT (CD45.1) and KO (Ncr1-iCre gp96fl/fl) donor bone marrow cells (5×106). NK cells were analyzed 8 weeks after reconstitution.
Cytolytic Assays
For cytolytic assays against YAC-1 cells, CFSE–labeled YAC-1 cells were cocultured with effector cells (mouse splenic NK cells) at the indicated effector:target (E:T) ratios for 4 hours. Next, cell mixtures were stained with 7-AAD to calculate killing efficiency as the percentage of 7-AAD+ YAC-1 cells with flow cytometry. To determine NK cell cytotoxic activity, 2 × 106 CFSE-labeled YAC-1 cells were intraperitoneally injected into WT and KO mice, respectively. Cells in the peritoneal cavity were harvested, and CFSE-labeled YAC-1 cells were measured 24 h post-injection.
Isolation of progenitor stem cells and NK differentiation in vitro
Bone marrow cells were obtained from WT or KO mice. Progenitor stem cells (PSCs) were enriched using an EasySep Mouse Progenitor Stem Cell Negative Selection kit (StemCell Technologies, Canada). Transduction was conducted immediately after the enrichment of PSCs using the spin protocol. PSCs (0.3 × 106) were placed into each well of a 48-well culture plate. After transduction, the virus-containing supernatant was removed. The transduced PSCs were cultured in NK differentiation conditioned medium39 (RPMI 1640 supplemented with 10% FBS, 1% PSG, 1.6 mmol/l 2-mercaptoethanol, 0.5 ng/ml of murine IL-7, 30 ng/ml of stromal cell factor, and 50 ng/ml FMS-related tyrosine kinase 3 ligand. On day 3, after transduction, 0.5 ml of fresh media was added. On day 5, cells were pelleted, old media was removed, and cultured in 0.5 ml of complete RPMI media containing 30 ng/ml of IL-15. On day 8, 0.5 ml of fresh media was added. On day 10, old media was replaced with fresh media containing 30 ng/ml of IL-15 and 1,000 U/ml of IL-2. On day 14, differentiated NK cells were analyzed by flow cytometry.
Virus Production And Transduction Protocols
Transduction of bone marrow progenitor cells was carried out as described previously 40. Briefly, cells were pelleted at 2,000 rpm for 5 min at room temperature in 1.5 ml screw-cap tubes and resuspended in 0.25 ml of viral supernatant (MOI = 10) in the presence of 8 µg/ml of Polybrene (Sigma, St. Louis, MO). Cells were incubated for 2 h at 37°C and 5% CO2. At the end of transduction, the virus-containing supernatant was removed.
BM NK differentiation in vitro
Bone marrow cells were cultured in RPMI1640 medium containing 10% fetal calf serum and 100 ng/ml stem-cell factor (SCF), with or without 50 ng/ml rIL15 (Genzyme). Flow cytometric and cytolytic analyses were done after culture for 10 days.
Tumor Models
Groups of six mice per experiment were used. The group size ensured enough power to determine biological differences. No mice were excluded in this study, and no active randomization was applied to groups. Single-cell suspension of MC38 colon cancer or LLC lung cancer cells was injected subcutaneously into the indicated strains of mice (2 × 105 cells per mouse). Mice were euthanized on days 21 to 28 following tumor injection for analysis of tumor-infiltrating lymphocytes. Tumor volumes were monitored with a caliper and calculated using the formula: V (in mm3) = 0.5 (ab2), where a is the longest diameter and b is the shortest diameter.
Isolation Of Tumor-infiltrating Lymphocytes (Tils)
TILs were isolated by dissociating tumor tissue in the presence of collagenase I (0.1% w/v, Sigma) and DNase (0.005% w/v, Sigma) for 1 h before centrifugation on a discontinuous Percoll gradient (GE Healthcare). After centrifugation, the white opaque layer at the interface between the two Percoll solutions, mostly containing leukocytes, is collected. Isolated cells were then used in various assays of NK cell function.
Intravenous Lung Metastasis Assay
For lung experimental-metastasis studies, 2.5 × 105 B16F10 cells were injected intravenously into WT or KO mice. The number of B16F10 melanoma surface nodules in the lungs of each mouse was counted. Two weeks after the injection, lung tumor nodules were counted.
Quantification And Statistical Analysis
FACS data were collected and processed using FACS software (FlowJo). GraphPad Prism 6 software was used to analyze data using a two-tailed paired Student’s t-test or two-way ANOVA. Data are represented as mean ± SD. P < 0.05 were considered significant.