Mice and human subjects
Female C57BL/6 mice were obtained from the Jackson Laboratory and maintained under specific pathogen-free conditions at the Duke University Animal Care Facility. Splenocytes were isolated from one 12-week-old mice for RNA extraction. All experiments involving animals were approved by the Duke University Institutional Animal Care and Use Committee (IACUC A128-20-06).
Peripheral blood mononuclear cells (PBMCs) were obtained from one human (Homo sapiens sapiens) subject under Duke Institutional Review Board Committee guidelines (IRB Pro00062495). Written informed consent was obtained.
Original plasmids and DNA templates for gene cloning
pU6-(BbsI)_CBh-Cas9-T2A-BFP (Addgene plasmid # 64323) was a gift from Ralf Kuehn30. pLV-EF1a-IRES-Puro (Addgene plasmid # 85132) was a gift from Tobias Meyer31. pLB (Addgene plasmid # 11619) was a gift from Stephan Kissler27. pMD2.G (Addgene plasmid # 12259) and psPAX2 (Addgene plasmid # 12260) were gifts from Didier Trono. mScarlet-I-mTurquoise2 (Addgene plasmid # 98839) was a gift from Dorus Gadella32. pLenti-smURFP-T2A-mCardinal (Addgene plasmid # 80348) was a gift from Erik Rodriguez and Roger Tsien33. pMito-miRFP703 (Addgene plasmid # 80000) was a gift from Vladislav Verkhusha15. pLSSmOrange-mKate2 (Addgene plasmid # 99868) was a gift from Marc Tramier34. Plasmid mNeonGreen-C1 was provided by Allele Biotechnology and Pharmaceuticals, Inc., under a license for non-commercial use.
DNA template coding FP hmKeima8.5 was synthesized (Genscript) based on published amino acid sequences17. Coding sequences for other FPs were cloned from plasmids mentioned above, with EBFP210 from pU6-(BbsI)_CBh-Cas9-T2A-BFP, mTurquoise211 from mScarlet-I-mTurquoise2, mNeonGreen12 from mNeonGreen-C1, mCardinal13 from pLenti-smURFP-T2A-mCardinal, mKate214 and LSSmOrange16 from pLSSmOrange-mKate2, and miRFP70315 from pMito-miRFP703. Coding sequences for cytoplasmic domain truncated mouse CD4 (amino acid (aa) 1–423), CD8a (aa 1–222), and CD86 (aa 1–268) were cloned from splenocyte cDNA samples from one C57BL/6 mouse. Coding sequences for full-length human CCR2b and CCR5, and cytoplasmic domain truncated human CD4 (aa 1–424), CD8a (aa 1–209), CD86 (aa 1–276), and CD154 (aa 1–240) were cloned from PBMC cDNA samples from one healthy donor.
DNA templates coding influenza HA antigens A/Christchurch/16/2010(H1N1) (GISAID Accession EPI280344) and A/Texas/50/2012(H3N2) (GenBank Accession KJ942744) were synthesized (Gene Universal, Inc.) based on amino acid sequences in the databases. Plasmid containing coding sequences for influenza HA A/Solomon Islands/3/2006(H1N1) (GenBank EU100724, with R540L correction) was provided by Aaron G. Schmidt (Ragon Institute). Plasmids containing coding sequences for other influenza HA antigens used were provided by Stephen C. Harrison (Harvard Medical School), including HA antigens A/Japan/305/1957(H2N2) (GenBank CY014976), A/Hong Kong/JY2/1968(H3N2) (GenBank CY147438), A/American black duck/New Brunswick/00464/2010(H4N6) (GenBank CY138045), A/Viet Nam/1203/2004(H5N1) (GenBank AY818135), A/Taiwan/2/2013(H6N1) (GISAID EPI459855), A/Taiwan/1/2017(H7N9) (GISAID EPI917065), A/northern shoveler/California/HKWF1204/2007(H8N4) (GenBank CY039588), A/Jiangxi/IPB13/2013(H10N8) (GenBank KJ406543), and A/mallard/Wisconsin/10OS3941/2010(H14N6) (GenBank CY133266).
Plasmid modification and gene cloning
Standard molecular cloning procedures were followed for plasmid modification and gene cloning. Endotoxin-free plasmids were prepared (E.Z.N.A.® Endo-free Plasmid DNA Mini Kit II, Omega Bio-tek) for mammalian cell transfection. Recombinant sequences in all plasmids used were verified by DNA Sanger sequencing (The Duke University DNA Analysis Facility).
The empty lentiviral transfer vector plasmids pLB-EF1a, pLB-EFS, and pLB-EF1a-IRES-Puro (pLB-EXIP; IRES stands for internal ribosome entry site) were constructed by replacing the U6-loxP-CMV-EGFP-loxP cassettes in plasmid pLB with EF1a promoter, EFS core promoter (nucleotide 1-226 of EF1a promoter, with attenuated transcription activity), or EF1a-IRES-Puro cassettes from plasmid pLV-EF1a-IRES-Puro, respectively. pLB-EXIP was further modified to generate bicistronic vectors pLB-EF1a-IRES-EBFP2, pLB-EF1a-IRES-mTurquoise2, pLB-EF1a-IRES-mCardinal, pLB-EF1a-IRES-mCD4, pLB-EF1a-IRES-mCD8a, and pLB-EF1a-IRES-mCD86 by replacing puromycin-resistant gene in pLB-EXIP with coding sequences for corresponding genes following IRES element. pLB-EFS-IRES-mNeonGreen was generated from pLB-EXIP by replacing EF1a promoter with EFS core protomer and puromycin-resistant gene with mNeonGreen coding sequences.
Lentiviral transfer vector plasmids expressing EBFP2, mTurquoise2, mCardinal, mKate2, miRFP703, LSSmOrange, and hmKeima8.5 were generated by cloning of corresponding coding sequences into pLB-EF1a. Lentiviral transfer vector plasmid expressing mNeonGreen was generated by cloning of mNeonGreen coding sequences into pLB-EFS. Human CD4, CD8a, CD86, and CD154 coding sequences (with cytoplasmic domain truncations, see above) were cloned into pLB-EXIP for puromycin-mediated selection. Human CD8a coding sequences were cloned into pLB-EF1a-IRES-mTurquoise2 and pLB-EF1a-IRES-mCardinal, and human CD86 coding sequences were cloned into pLB-EF1a-IRES-EBFP2 and pLB-EFS-IRES-mNeonGreen. These bicistronic expression vectors can co-express human CD8a/CD86 and FPs, with FPs at a lower expression level driven by IRES element. Coding sequences for influenza HA antigens, human CCR2b, CCR5, and their domain-swapped or point mutants were cloned into pLB-EXIP.
Culture, transfection, and transduction of mammalian cell lines
HEK 293T and K562 cell lines were purchased from ATCC. HEK 293T cells were cultured in Dulbecco’s modified Eagle’s medium (Gibco) supplemented with 10% heat-inactivated HyClone fetal bovine serum (FBS) (Cytiva), 10 mM HEPES buffer, and 55 µM 2-Mercaptoethanol (all Gibco). K562 and derivative cell lines were maintained in RPMI-1640 medium (Gibco) supplemented with 10% heat-inactivated HyClone FBS, 10 mM HEPES buffer, 1 mM sodium pyruvate, 1× MEM NEAA, 55 µM 2-Mercaptoethanol, 100 units/ml penicillin, and 100 µg/ml streptomycin (all Gibco). For all K562-derivative cell lines, monoclonal cell lines were established by single-cell sorting (see below) and used in binding assays in this study.
Single-guide RNAs (sgRNAs) targeting human CD32A exon 1 were designed with the online tool (http://crispr.mit.edu). sgRNAs used in this study were sgRNA-hCD32A-1 (5′-AGCAGCAGCAAAACTGTCAA-3′), sgRNA-hCD32A-2 (5′-ATGTATGTCCCAGAAACCTG-3′), and a negative control sgRNA (5′-TGTCATGCGTCACTTAGTGC-3′). Corresponding DNA oligos were synthesized and cloned into plasmid pU6-(BbsI)_CBh-Cas9-T2A-BFP. K562 cells were transfected with CRISPR-Cas9 targeting plasmids using Lipofectamine 3000 Transfection Reagent (Invitrogen). Ninety-six hours after transfection, the cells were collected for flow cytometry analysis and single-cell sorting (see below).
Lentiviral transfer vector plasmids were co-transfected into HEK 293T cells with packaging plasmids pMD2.G and psPAX2 using Lipofectamine 3000 Transfection Reagent (Invitrogen). Forty-eight hours after transfection, culture supernatants were collected and filtered through 0.45 µm polyvinylidene difluoride membrane filters (Millipore). K562-derivative cell lines were transduced with the filtered supernatants containing lentiviral vectors by spinoculation at 1000 × g for 45 min at 32 °C. For transductions with pLB-EXIP-based vectors, cells were selected with puromycin (Sigma, 2 µg/ml) between 3 and 7 days after transduction. Seven days after transduction, the cells were collected for flow cytometry analysis and single-cell sorting (see below).
Antibodies
Monoclonal antibodies used in this study included the following: FITC-conjugated anti-human CD32A (hCD32A-FITC, clone IV.3, STEMCELL 60012FI), hCD32-APC (clone FLI8.26, BD 559769), hCD16-PE (clone 3G8, BioLegend 302007), hCD32-PE (clone FLI8.26, BD 550586), hCD64-PE (clone 10.1, BioLegend 305007), hCD4 (clone SK3, BioLegend 344602), hCD8a (clone HIT8a, BioLegend 300902), hCD86 (clone BU63, BioLegend 374202), hCD154 (clone 24-31, BioLegend 310802), mCD8a-PerCP-eFluor710 (mouse CD8a, clone 53-6.7, ThermoFisher 46-0081), mCD86-PE-Vio770 (clone PO3.3, Miltenyi Biotec 130-105-135), mCD4-APC-Fire750 (clone RM4-5, BioLegend 100568), hCD58-PE (clone MEM-63, ThermoFisher MA1-10256), HA-tag (clone 16B12, BioLegend 901533), hCCR2 (clone K036C2, BioLegend 357201), hCCR2 (clone 48607, R&D Systems MAB150), hCCR5 (clone 2D7, BD 555991), hCCR5 (clone CTC8, R&D Systems MAB1801), hCCR5 (clone 45523, R&D Systems MAB181), hCCR5 (clone 45529, R&D Systems MAB184), and hCCR5 (clone 45549, R&D Systems MAB183). Isotype control antibodies included the following: Mouse IgG1 κ-isotype control (Rockland 010-001-330), Human IgG1κ (hIgG1K, Southern Biotech 0151K-01), and Human IgG1 Lambda (hIgG1L, Southern Biotech 0151L-01). Secondary antibodies included the following: Goat Anti-Human IgG-PE (Southern Biotech 2040-09), Goat Anti-Mouse IgG, and Human ads-PE (Southern Biotech 1030-09). Influenza HA-specific antibodies FI618, S5V2-2919, CH6720, and HC1921 were prepared as recombinant human IgG1 antibodies as described35.
Cell surface staining, flow cytometry analysis, and single-cell sorting and cloning
Cultures of K562 and derivative cells were collected, centrifuged at 300 × g for 2 min at 4 °C, and resuspended in staining buffer (phosphate-buffered saline supplemented with 2% heat-inactivated FBS). After incubation with antibodies at 4 °C in the dark for 30 min, cells were washed with staining buffer and resuspended in staining buffer for either secondary staining following the same procedure above or stored on ice for flow cytometry analysis or single-cell sorting. For cell counting, CountBright™ Absolute Counting Beads (Invitrogen) were added into cell suspensions prior to flow cytometry analysis.
Flow cytometry analysis was carried out using either BD FACSCanto II cytometer (Duke Cancer Institute Flow Cytometry Shared Resource) or BD LSR II cytometer (The Duke Human Vaccine Institute (DHVI) Research Flow Cytometry Facility). Single-cell sorting was performed with BD Aria II (The DHVI Research Flow Cytometry Facility). The bulk cell line after transfection or transduction were stained with corresponding antibodies and single cells expressing FP or antigen of interest were sorted into 96-well flat-bottom plates containing 100 µl/well of the complete RPMI medium above supplemented with 20% heat-inactivated FBS. Nine days after sorting, healthily proliferating cell clones were transferred into 24-well plates for further expansion. Three days later, individual monoclonal cell lines were validated for the expression of FP or antigen of interest and a single clone with a uniform expression level was selected for further engineering or used as a reporter cell line in immunoassays.
Data analysis
FlowJO (Version 10.7.2), Chromas (Version 2.6.6) and Microsoft Office (Version 1808) were used to analyze data and prepare figures for publication.
Statistics and reproducibility
The reproducibility of flow cytometry detections of the expression of FPs and cell surface molecules was confirmed by two or three independently repeated experiments as indicated in figure legends and shown as Supplementary Figures. The proliferation rates of reporter cell lines were determined with two or three independent cultures for each time point as indicated in corresponding figure legends.
Reporting summary
Further information on research design is available in the Nature Research Reporting Summary linked to this article.
