Material and Methods
Ethics Statement
Work performed with primary cells isolated from blood donations was
invariably obtained from anonymous donors that had provided written
informed consent in full compliance with the requirements of the Ethics
Committee of the University Hospital Frankfurt, Germany.
Cell lines and primary
cells
HEK293T (ATCC CRL-11268), HT1080 (ATCC CCL-121) and
HT1080αHis cells were cultivated in DMEM (Sigma-Aldrich,
Munich, Germany) supplemented with 10% fetal calf serum (FCS; Biochrom,
Berlin, Germany) and 2 mM L-glutamine (Sigma-Aldrich, Munich, Germany).
Culture medium of HT1080αHis cells was furthermore
supplemented with1.2 mg/mL G418 (Thermo Fisher Scientific, Darmstadt,
Germany). The cell line HT1080CD62L was generated by LV
transduction of the parental HT1080 cell line with LV particles
packaging a construct encoding a spleen focus-forming virus (SFFV)
promoter, the gene encoding CD62L (UniProt: P14151), an internal
ribosome entry site (IRES) element followed by a puromycin resistance
gene and a woodchuck posttranscriptional regulatory element (WPRE)
(transfer plasmid: pS-CD62L-IPW). Transduced cells were selected using
puromycin for 2 weeks. Nalm-6-eBFP-Luc (kindly provided by Prof. Helen
Fielding, University College of London), further on called Nalm6, were
grown in complete Roswell Park Memorial Institute (RPMI) medium (RPMI
1640, Biowest), supplemented with 10% FCS and 2 mM L-glutamine.
Human PBMC were isolated from fresh blood of healthy donors or buffy
coats purchased from the German blood donation center
(DRK-Blutspendedienst Hessen, Frankfurt) and cultured in T cell medium
(TCM) consisting of RPMI 1640 supplemented with 10% FCS, 2 mM
L-glutamine, 0.5% streptomycin/penicillin, 25 mM HEPES (Sigma-Aldrich,
Germany), 25 U/mL IL-7 and 50 U/mL IL-15 (all cytokines from Miltenyi
Biotec, Germany). For activation, 1x107 PBMC per
6-well were cultured in TCM supplemented with 3 μg/mL anti-CD28 antibody
(clone 15E8, Miltenyi Biotec, Germany) for 48 hours. 6-well plates for
activation were pre-coated with 1 µg/mL anti-CD3 antibody (clone OKT3,
Miltenyi Biotec, Germany).
Generation of CD62L-targeted envelope
constructs
To generate the CD62L targeting constructs, the coding sequences of the
variable light chain (VL) and heavy chain (VH) of the parental
CD62L-specific monoclonal antibody 145/15 were synthesized de novo
(GeneArt, Thermo Fisher Scientific) and cloned into the backbone
encoding the modified Nipah virus (NiV) glycoprotein G with and without
glycine-serine linker (pCG-GNiVΔ34mut-His and
pCG-GNiVΔ34mut-L3-His) or the modified measles virus
(MV) hemagglutinin protein (pCG-HMVnseΔ18mut-L3-His) via
digestion with Sfi I and Not I. DNA sequences were verified
by standard sequencing technologies prior to use in LV production.
LV production and characterization
Vector particles were generated by transient transfection of HEK-293T
cells using polyethylenimine (PEI) and second generation packaging
plasmids as described in detail by Weidner and colleagues. In brief, one
day before transfection, 1.5-2x107 cells were seeded
into a T175 flask. In total, 35 μg DNA was added to 2.3 mL of DMEM
without additives and combined with 2.2 mL DMEM containing 140 μL of 18
mM PEI solution. The transfection solution was mixed and incubated for
20 min at room temperature. The cell medium was replaced by 10 mL DMEM
supplemented with 15% FCS and 3 mM L-glutamine, before the transfection
solution was added to HEK-293T cells. 4-6 hours later the medium was
replaced by DMEM with 10% FCS and 2 mM L-glutamine. Two days after
transfection, the cell culture supernatant was collected and filtrated.
Released vector particles were concentrated over a 20% sucrose cushion
at 4500xg for 24 hours, before supernatant was discarded and pellets
were resuspended in 60 µL PBS per T175 flask. The used transfer plasmid
encoded a second generation CD19-CAR in conjunction with ΔLNGFR.
Notably, based on the co-expression of ΔLNGFR and the CAR construct,
detection of ΔLNGFR can be used as surrogate marker for the expression
of CAR molecules on the cell surface. Plasmid ratios for the generation
of NiV-based and MV-based RT-LV particles as well as particles
pseudotyped with VSV-G were described previously and can be found in the
supplemental table 1. If not otherwise specified, all concentrated
vector stocks were titrated on HT1080αHis cells as
described previously using a LNGFR specific antibody for detection. LV
particle yields were determined by nanoparticle tracking analysis or
p24-specific enzyme-linked immunosorbent assay (HIV type 1 p24 Antigen
ELISA; ZeptoMetrix Corporation) according to the manufacturer’s
instructions and calculated as described.
Transduction of cell lines and primary
cells
Parental HT1080, HT1080αHis, and
HT1080CD62L cells were seeded at 8x103cells per 96-well and incubated with serial dilutions of vector stocks.
Transgene expression was analyzed 72 to 96 hours later by flow
cytometry. Activated PBMC were seeded at 4x104 or
8x104 cells per 96-well, respectively, in TCM medium
before CD62L-LV (5 µL or 10 µL) or VSV-LV (0.05 µL or 0.5 µL) were
added. Where indicated, CD62L-LV transduction of PBMC was carried out in
presence of Vectofusin-1 (Miltenyi Biotec, Germany) as described
previously. Cells were centrifuged at 850g, 32°C for 90 minutes,
followed by addition of TCM. Medium was replenished every 2 to 3 days.
Transgene expression was assessed by flow cytometry.
Quantification of shed CD62L by
ELISA
Activated PBMC of three donors were cultured without medium change or
cell passaging for up to 10 days. At the indicated time points, cell
suspension was collected and centrifuged for 5 minutes at 5,000 rpm and
either stored at -80°C or 4°C. Concentration of sCD62L in supernatant
was determined by ELISA (Human L-Selectin/CD62L DuoSet ELISA, R&D
Systems) following the manufacturer’s protocol with the exception that
heat inactivated FBS was used instead of inactivated goat serum.
Quantification of the fluorescent signals was performed with a
microplate reader (EmaxPlus, Molecular Devices).
Blocking assay with shed CD62L or
antibodies
For the blocking assay with antibodies, 4x104activated PBMC were preincubated with the indicated concentrations of a
CD62L-specific antibody (clone 145/15, Miltenyi Biotec) or a
CD45-specific antibody (clone 5B1, Miltenyi Biotec) either conjugated to
the fluorophore phycoerythrin (PE)-Vio770 or to biotin for 1 h at 4°C.
Before and after antibody incubation, cells were washed twice with wash
buffer (phosphate-buffered saline (PBS) supplemented with 2% FBS and
0.1% NaN3). Afterwards, either 10 µL of 62L-LV or PBS
were added to cells pre-incubated with biotin conjugated antibodies,
while PBS was added to cells pre-incubated with fluorophore-conjugated
antibodies. All samples were incubated at 4°C for 30 min. Cells
pre-incubated with biotin conjugated antibodies were further stained
with a PE-labeled anti-LNGFR antibody (clone ME20.4-1.H4, Miltenyi
Biotec). After two additional washing steps, antibody and vector bound
cells were determined by flow cytometry analysis respectively.
For the sCD62L blocking assays, 10 µL of 62L-LV or VSV-LV vector
particles were pre-incubated with 90 µL fresh or frozen supernatant
containing sCD62L derived from six days of PBMC culture or TCM only for
1 h at 4°C. Vector/sCD62L-containing supernatant was then added to
4x104 activated PBMC of various donors in 96-wells.
Staining for vector bound cells was performed after an incubation for 30
minutes at 4°C by flow cytometry detecting ΔLNGFR.
Animal experiment
All animal experiments were conducted in accordance with the German
Animal Protection Law and the respective European Union guidelines.
For short-time generation of CAR T cells, 1.8x106activated PBMCs were seeded in 600 µL TCM per 24 well, mixed with 30.6
µL 62L-LV (equals MOI of 1.3 or
~4x1010 particles) or VSV-LV (equals
an MOI of 8.8 or ~3x1010 particles) or
equal volume of PBS and centrifuged for 90 min at 850g, 32°C before
addition of TCM to a total volume of 1.2 mL per well. 24h after vector
incubation, cells were harvested and washed 2x with PBS prior in vivo
application. NSG mice
(NOD.Cg.PrkdcscidIL2rgtmWjl/SzJ,
Jackson Laboratory) were intravenously (i.v.) injected with
2x106 vector bound cells or 1.4x106PBS treated cells. Three days later, 5x105 Nalm-6 were
injected i.v. and tumor growth was followed by bioluminescence imaging
(BLI). This was performed by injecting D-luciferin (Perkin Elmer)
intraperitoneally at 150 µg/kg body weight and imaging luciferase
signals 10 minutes after injection using the IVIS Imaging System (Perkin
Elmer). CAR T cell engraftment was monitored through regular, blood
drawings. Mice were checked regularly for health status and tumor load
by IVIS. All mice were sacrificed on day 17 for final analysis of blood
and organs (spleen, bone marrow and liver).
Collected blood and organs were prepared to a single cell suspension and
analyzed by flow cytometry analysis. Blood was washed with PBS prior and
after erythrocyte lysis using BD Pharm Lyse buffer (BD Bioscience).
Spleens were minced through a 70 µm cell strainer to obtain a single
cell solution and proceeded with erythrocyte lysis. Bone marrow was
harvested through centrifugation of long bones cut open with a scalpel
in pierced 0.5 mL tubes at 8000 rpm for 5 min. Bone marrow cells were
then washed with PBS and singularized through a 70 µm cell strainer and
erythrocyte lysis was performed. Liver cells were isolated using the
mouse liver dissociation kit (Miltenyi Biotec) according to the
manufacturer’s instruction, washed with PBS and erythrocytes were lysed.
Flow cytometry
Flow cytometry analysis was performed using MACSQuant Analyzer 10
(Miltenyi Biotec, Bergisch Gladbach, Germany) or LSR Fortessa (BD
Biosciences) flow cytometers. Data were analyzed by FCS Express 6 (De
Novo Software, Glendale, CA, USA) or FlowJo 7 (BD Biosciences). Before
and after staining with fluorescently labelled antibodies, cells were
washed twice with wash buffer. Before measurement cells were fixed by
addition of PBS supplemented with 1% formaldehyde. To determine the
percentage of transduced cells or cell-bound vector particles, staining
of up to 1x105 cells was performed. The reporter
protein ΔLNGFR, which is co-expressed with the CD19-CAR, was detected
using the anti-LNGFR-PE antibody. PBMC were further stained with the
fixable viability dye eFluor780 (Life Technologies, Darmstadt, Germany),
according to the manufacturer’s instructions or with 7-AAD to detect
viable cells. To further characterize the PBMC, cells were stained in
addition with a CD4-specific antibody (clone VIT4) labeled with VioGreen
or PE-Vio770 and a CD8-specific antibody (clone BW135/80) labeled with
allophycocyanin (APC) or APCVio770 and if indicated with a
CD62L-specific antibody (clone 145/15) labeled with PEVio770. All
antibodies were from Miltenyi Biotec (Bergisch Gladbach, Germany). The
following antibodies were used for flow cytometry analysis of thein vivo experiment: CD45-BV510 (clone 2D1, BioLegend), CD3-BV605
(clone HIT3a, BD Bioscience), CD8-BV786 (clone RPA-T8, BD Bioscience),
LNGFR-PE (clone ME20.4-1.H4, Miltenyi Biotec), CD4-PE-CF594 (clone
RPA-T4, BD Bioscience), CD19-Alexa Fluor 700 (clone HIB19, Thermo
Fisher), eFluor780 (eBioscience).
Statistical Analysis
Statistical analyses were performed with Prism 7 software (GraphPad).
Tests for statistical significance used the unpaired or paired
two-tailed Student’s t test, one-way ANOVA (Dunnett multiple comparisons
test), two-way ANOVA (Dunnett or Turkey multiple comparisons test) or
Fisher’s least significant difference (LSD) test as indicated.
Statistical differences in experiments were considered significant atp < 0.05.