Figure Legends
Figure 1: Basic characterization of 62L-LV.
A) Physical properties of 62L-LV vector stocks. Three
independently produced 62L-LV stocks were analyzed for size (left panel)
and for particle concentration (right panel) by nanoparticle tracking
analysis (filled bar; technical triplicates) or p24-ELISA (open bar,
biological replicates). Means and standard deviations (SD) are depicted.B ) 62L-LV stocks were titrated on HT1080αHiscells or activated human PBMC. Individual results and means with
standard error (SEM) are plotted. Statistical significance was
determined by using unpaired t-test. C ) Target and non-target
HT1080 cells were incubated with 2.5 µL 62L-LV stock for 4 days or left
untransduced. Antibody staining for ΔLNGFR allowed detection of
transduced cells by flow cytometry. Left panel: Representative dot plots
of one vector stock. Right panel: Percent ΔLNGFR expressing HT1080 cells
after transduction with seven different vector stocks. Dashed lines
indicate baseline ΔLNGFR levels on each individual cell line. Individual
results as well as means with SD are plotted. Statistical testing was
performed by using ordinary 1-way ANOVA. WT = parental HT1080 cells.
Figure 2: CAR gene delivery to primary lymphocytes by
62L-LV
Activated PBMC were incubated with 62L-LV (blue dots) or VSV-LV (orange
dots). A) Transduction rates in the presence (+V1) or absence
(-V1) of Vectofusin-1. Left panel: Representative dot plots of 62L-LV
transduced PBMC pre-gated for CD3+ cells. Right panel: Results for PBMC
from seven different donors in four independent experiments analyzed 9 -
12 days post-transduction. For VSV-LV, no V1 was applied. Individual
results and means with standard deviation (SD) are plotted. Statistical
analysis was performed by using paired t-test for the comparison of
62L-LV +/-V1 and by using unpaired t-test for the comparison of 62L-LV
and VSV-LV. B) The total percentage of ΔLNGFR+ cells expressing CD62L is
displayed for the CD4+ (left) and CD8+ (right) fractions. Transduction
was performed in absence of V1 for 62L-LV and VSV-LV, respectively.
Cells transduced with either vector were tested for significant
differences at each analysis time point individually by Fisher’s least
significant difference (LSD) test. Mean with standard error (SEM) of 8
individual experiments is plotted.
Figure 3: Selectivity in 62L-LV binding to primary T
lymphocytes .
Activated PBMC incubated either with the CD62L-specific antibody (blue
bars) or with the CD45-specific antibody (grey bars) at the indicated
concentrations before PBS (open bars) or 62L-LV vector particles (filled
bars) were added for 30 min at 4°C. A ) Fluorophore-labelled
αCD62L and αCD45 antibodies were used to determine the staining
intensity of CD62L and CD45 on activated PBMC at the indicated
concentrations by flow cytometry. Mean fluorescent intensities (MFI) are
shown. B ) PBMC were pre-incubated with biotin-labelled
antibodies before addition of 62L-LV particles. After vector incubation,
cells were stained with fluorophore coupled αCD3 and αLNGFR antibodies
to allow detection of vector bound T cells by flow cytometry. Background
MFI (dashed line) is determined from samples incubated with PBS (w/o)
instead of 62L-LV accumulated for all antibody concentrations. Means of
MFI and standard deviations (SD) are depicted. Statistical testing was
performed by using 2-way ANOVA.
Figure 4: Shed CD62L does not influence vector binding.
A ) Accumulation of sCD62L in the supernatant of PBMC. Frozen
PBMC were thawed, activated and cultivated in presence of IL-7 and
IL-15. The complete supernatant of one well was collected at the
indicated day and used for sCD62L quantification (left panel). The
amounts of sCD62L present in three independent cultures at day 6 are
shown in the right panel. Individual results, means and standard
deviation (SD) are depicted. B ) 62L-LV (blue) or VSV-LV
(orange) particles were incubated with fresh or frozen supernatant
containing sCD62L (day 6 harvest) or cell medium (TCM) only. Mixture of
vector stock and supernatant was incubated with activated PBMC for 30
min at 4°C. Flow cytometry was performed analyzing content of vector
bound T cells by staining with fluorophore coupled αCD3 and αLNGFR
antibodies. Dot plots of vector bound T cells are depicted in the right
panel. Percentage of vector bound cells is indicated. ΔLNGFR intensity
[MFI] of vector bound cells in 3 - 10 independent experiments is
shown in the left panel. Individual results and means with SD are
plotted. Statistical testing was performed by using 2-way ANOVA.
Figure 5: Antitumoral activity of CAR T cells generated
with
62L-LV.
A) Experimental setting. PBMC were activated for two days prior
to 24h incubation with 62L-LV, VSV-LV or PBS (control) and injected i.v.
into NSG mice. Nalm6 cells were injected at day 4 post adoptive cell
transfer and their growth was followed by bioluminescence imaging (BLI).B) Monitoring for tumor load by BLI at the indicated days after
adoptive cell transfer. Ventral images of each mouse are depicted.C) Total body flux quantified at the indicated time points for
the 62L-LV group (blue), the VSV-LV group (orange) and the control
(grey). Mean with standard error (SEM) is plotted. Dotted line
represents background signal of mice without imaging substrate. Ordinary
1-way ANOVA was used to determine statistics. D) Cells isolated
from blood and organs of each mouse were analyzed by flow cytometry for
viable, CD45 negative, CD19 and EBFP double-positive Nalm6 cells. The
percentage of Nalm6 positive cells of all viable cells is depicted.
Individual results and means with standard error are plotted.
Figure 6: Expression of ΔLNGFR+ in human
CD4+ or CD8+ T cells
A) Presence of ΔLNGFR+ cells on human CD4+ or
CD8+ T cells in blood determined by flow cytometry on
day 3, 10 and 17 of the experiment. Gating on viable human
CD45+, CD3+ and respective lineage
marker positive cells was performed. Only data with at least 20 events
in CD4+ or CD8+ T cell gate are
shown. Mean with standard error (SEM) are depicted. B-E)Presence of human CD45+ cells (left), ΔLNGFR+ human
CD4+ (middle) or ΔLNGFR+ human CD8+T cells (right) in spleen (B ), blood (C ), bone marrow
(D ) and liver (E ) as determined by flow cytometry at
final analysis. Individual results and mean with standard deviation (SD)
are depicted. Unpaired t-tests were performed to determine statistics.