Bacterial strains, growth conditions, and plasmid
Lactococcus (L.) lactis subsp. cremoris NZ9000 (NZ9000) was purchased from MoBiTec GmbH (Gottingen, Germany). NZ9000 is a derivative of L. lactis subsp. cremoris MG1363 in which the pepN gene has been replaced with the constituent genes of the NICE system, i.e., nisR and nisK (Ruyter et al. 1996). NZ9000 was cultured in M17 broth (BD Difco™, Becton, Dickinson and Co., MD, USA) containing 0.5% glucose (GM17) at 30 °C without shaking. Constructed gmLAB were cultured using GM17 supplemented with 10 µg/mL chloramphenicol (GM17cm). Escherichia (E.) coli MC1061 was purchased from MoBiTec GmbH and cultured using Luria-Bertani (LB) broth (Invitrogen Corp., CA, USA) containing 25 µg/mL chloramphenicol (LBcm) at 37 °C with intense shaking.
The NICE-system plasmids, pNZ8148#2:CYT and pNZ8148#2:CYT-GFP, were constructed based on the commercially available pNZ8148 (MoBiTec GmbH), as described in Shigemori et al., and were used as the gene expression vectors (Fig. 1a, b) (Shigemori et al. 2012, 2017b).
Design of the scFv and construction of the gene expression vector
PDL1scFv was designed by connecting the amino acid sequences of the variable region of atezolizumab using the flexible peptide linker EGKSSGSGSESKS. The three-dimensional (3D) structure of the designed PDL1scFv was predicted using SWISS-MODEL, an automated protein homology-modeling server (Schwede et al. 2003). The designed amino acid sequences were then converted to nucleotide sequences by Eurofins Genomics (Tokyo, Japan), based on L. lactis subsp. cremoris MG1363 codon usage. In addition, restriction enzyme recognition sites, BamHI and HindIII, were inserted on each side of the scFv sequence. The resulting gene was subcloned into pEX-K4J2 by Eurofins Genomics (Tokyo, Japan). General molecular cloning techniques were performed using modifications of previously described methods (Namai et al. 2018a). Briefly, the gene segment was excised using BamHI and HindIII and cloned into the multi-cloning site of pNZ8148#2:CYT-GFP. The resulting GFP-conjugated PDL1scFv (GFP-PDL1scFv) expression vector (designated pNZ8148#2:CYT-GFP-PDL1scFv) was sequenced by Eurofins Genomics (Tokyo, Japan) to confirm the absence of mutations and/or deletions.
Construction of gmLAB for GFP-PDL1scFv gene expression
pNZ8148#2:CYT-GFP-PDL1scFv (DDBJ accession number: LC739557) was introduced into NZ9000 to construct a gmLAB strain (designated as NZ-GFP-PDL1scFv) by electroporation (Namai et al. 2018a). Simultaneously, pNZ8148#2:CYT and pNZ8148#2:CYT-GFP were also introduced into NZ9000 to generate the vector control gmLAB (designated as NZ-VC and NZ-GFP). The constructed gmLAB were then cultured to induce gene expression (Namai et al. 2018b). Briefly, the pre-incubated gmLAB were inoculated into GM17cm (final concentration 5%), and nisin, a gene expression inducer, was added when the optical density at 600 nm (OD600) reached 0.4 (1-1.5 h). Cells were harvested at 3 h after the addition of nisin by centrifugation (4 °C, 8,000×g, 5 min) and washed with ice-cold Tris-buffered saline (TBS: 50 mM Tris-HCl, 140 mM sodium chloride, pH 8.0) or phosphate-buffered saline (PBS; 137 mM sodium chloride, 2.7 mM potassium chloride, 10 mM disodium hydrogen phosphate, 1.76 mM potassium dihydrogen phosphate, pH 7.4). Then, the cell pellets were crushed using a bead beater (µ-12; TITEC, Saitama, Japan), and the soluble fraction was obtained by centrifugation (4 °C, 20,000×g, 15 min). An equal volume of 2× sample buffer (Wako, Osaka, Japan) was added to the soluble fraction and boiled at 95 °C for 5 min to prepare the sample for western blotting (WB) (Ishida et al. 2020; Namai et al. 2018a).
Confocal laser scanning microscopy
Freshly prepared TBS-washed bacterial pellets were suspended in 400 µL of TBS, and 10 µL was placed on a microscope slide and observed under a confocal laser scanning microscope (FluoView FV1000, Olympus, Tokyo, Japan) using an oil immersion objective lens (×60).
Immunoreactivity assay of GFP-PDL1scFv
Recombinant gene expression was induced, and the cell pellet was crushed to obtain a soluble fraction containing recombinant protein, as described above. The total protein concentration of the soluble fraction was measured using a BCA Protein Assay Kit (Thermo Fisher Scientific, MA, USA) according to the manufacturer’s instructions, and the solution was adjusted to 5 mg/mL. The immunoreactivity of the recombinant GFP-PDL1scFv (rGFP-PDL1scFv) was examined using an enzyme-linked immunosorbent assay (ELISA), as described previously (Namai et al. 2020a, b; Shigemori et al. 2017a).
Purification of rGFP-PDL1scFv
Recombinant gene expression was induced by adding nisin (final: 1.25 ng/mL) to 1 L culture, as described above. The cell pellet was then collected by centrifugation (4 °C, 8,000×g, 5 min), washed using MilliQ water, and frozen at −80 °C. The frozen pellets were pulverized into a fine powder in liquid nitrogen using a Cryo Press disruptor (Microtec Co., Chiba, Japan), followed by adding 10 mL binding buffer (20 mM imidazole, 20 mM Na3PO4, 0.5 M NaCl, pH 7.4). The soluble fraction was collected (4 °C, 12,000×g, 15 min), and DNA was removed using a Nucleic Acid Removal Kit (ProFoldin, Hudson, MA, USA) as per the manufacturer’s instructions. The resulting fraction was filtered using a DISMIC-25AS filter (pore size 0.45 μm, Toyo Roshi, Tokyo, Japan). The filtrate was loaded onto a HisTrap HP column (1 mL, GE Healthcare) equilibrated with binding buffer, and the column was washed with five column volumes (CV) of binding buffer. The column-absorbed proteins were then eluted with a linear gradient of 0–500 mM imidazole over 40 CVs at 1 mL/min using a fast protein liquid chromatography system (AKTA pure 25, GE Healthcare). The collected fractions (soluble fraction; cell, wash, flow-through, and eluate; F1-8) were analyzed by WB with CBB staining following SDS-PAGE, as described above. The eluted fractions were then dialyzed against PBS, and the His-tagged protein concentration in the dialyzed sample was measured using a His-Tag ELISA Detection Kit (GenScript, Piscataway, NJ, USA).
Culture conditions for Raw264.7 cells
The mouse macrophage cell line, Raw264.7 cells (ATCC, Manassas, VA, USA), was maintained in complete Dulbecco’s Modified Eagle’s medium (DMEM; with 10% fetal bovine serum [GE Healthcare], penicillin [100 U/mL; Nacalai Tesque, Kyoto, Japan], and streptomycin [100 µg/mL; Nacalai Tesque]) at 37 °C in 5% CO2 and passaged once every three days.
Flow cytometry of Raw264.7 cells
Raw264.7 cells were seeded on a 24-well plate at 2.0 × 106 cells/well and cultured at 37 °C for 2 h. After removing the supernatant, a complete DMEM containing 0 or 10 µg/mL of lipopolysaccharide (LPS; InvivoGen, San Diego, CA, USA) was added. After 4 h of incubation at 37 °C, cells were collected by centrifugation (4 °C, 500×g, 5 min) and washed with PBS containing 1% fetal bovine serum. Cells were then stained using 1/100 dilution of PE anti-mouse CD274 (B7-H1, PD-L1) Antibody (BioLegend, San Diego, CA, USA) or purified rGFP-PDL1scFv (500 ng/mL) at RT for 1 h. After washing, cells were analyzed using a Cell Sorter SH800 (SONY, Tokyo, Japan), and cell populations were identified using FlowJo software (v10.5.3; BD Biosciences, San Jose, NJ, USA).
Fluorescence observation of Raw264.7 cells
Raw264.7 cells were seeded on a 24-well plate at 2.0 × 106 cells/well and cultured at 37 °C for 2 h. After removing the supernatant, a complete DMEM containing 0 or 1 µg/mL of LPS was added. After 24 h incubation at 37 °C, the supernatant was removed, and cells were fixed using 200 µL of 10% formalin neutral buffer solution for 10 min. Then, cells were washed twice with PBS containing 0.05% Tween 20 (Nacalai) (PBS-T) and stained using 200 µL of 1/100 dilution of PE anti-mouse CD274 (B7-H1, PD-L1) antibody or purified rGFP-PDL1scFv (500 ng/mL) for 1 h. Cells were washed twice using PBS-T and mounted using DAPI-Fluoromount-G (Southern Biotech, Birmingham, AL, USA). The resulting slides were observed under a BZ-X800 microscope (Keyence, Osaka, Japan).
Statistical analysis
GraphPad Prism software (version 8, GraphPad, San Diego, CA, USA) was employed for statistical analysis, and significance was accepted at p < 0.05. For flow cytometry analysis, the data were analyzed using unpaired t-tests.