Figure 1.
Enhanced gene transfer after direct cell-cell contact of 1o and 2o targets.
(A) Experimental design, illustrating cell-free (left) and cell-bound (right) transduction strategies. (B) Ratio of relative infectivity (% GFP / ng p24) after 24-hour direct cell-free transduction versus transduction by (Jurkat) cell-bound particles following co-culture. The y-axis ratio reflects GFP marking (FACS) per ng input vector (p24 ELISA) at increasing vector particle numbers (x-axis), corresponding to MOI 0.01-3. Refer to Methods section for detailed procedure. (C) Residual infectivity of cell surface bound versus cell-free particles. Particles were kept at 37oC and identical aliquots (MOI 1, based on vector titer) were removed at indicated time points for transduction culture of 1 x105 293T cells (cell free, black circles). For the cell-bound conditions, the same number of vector particles were arrested on the surface of 1o target Jurkat cells (MOI 1) at 4oC, before shift to 37oC. Aliquots of 1 x105 cells were subsequently placed in coculture with 293T 2o targets at the indicated time-points. A 2-tailed paired Student’s t test was performed; p values ≤ 0.01 are indicated by double asterisks. (D) Relative GFP marking in secondary 293T cells after direct co-culture (24 hours) by escalating the number of GFP vector exposed Jurkat cells (1o, CD45+) to 293T (2o, CD45-) cells at stable MOI of 1 (ratio, gray bars), or by escalating MOI during exposure of primary cells, with matched 1:1 cell numbers (MOI, red bars). Direct transduction of 293T cells with cell-free vector (black bar, control). (E) Illustration of experimental design used in (F). (F) Transduction of Jurkat cells under high cell density transduction (1x106 cells per ml, black circles) or low cell density (1x105 cells per ml, open circles) transduction conditions over a range of MOI. Cells were transduced in the presence of 4 µg/ml protamine sulfate. Following a 3-hour transduction at 37°C, cells were washed, placed back in culture at 37°C, and flow cytometry was performed 72 hours later. All experiments were repeated with similar results.
Figure 2.
VSV-G pseudotyped lentivector particles do not form aggregates.
(A) GFPvpr labeled vector particles were allowed to settle on glass slides in the absence (top) or presence (bottom) of recombinant fibronectin fragment, CH296, stained with anti-VSV-G antibody and Alexa Fluor 647 anti-mouse secondary antibody, and visualized via deconvolution microscopy. (B) Particles either counted as “individual” (gray bars) or as “aggregate” (black bars) if 2 or more individual particles were observed to occupy contiguous space. Significance was determined by performing a Student’s 2-tailed t-test, assuming unequal variance between groups. (C) GFPvpr labeled particles were allowed to settle on glass slides in the absence (top) or presence (bottom) of 8 µg/ml protamine sulfate. (D) Particles either counted as “individual” (gray bars) or as “aggregate” (black bars) if 2 or more individual particles were observed to occupy contiguous space. Asterisk indicates that data set for “(-PS) control is the same from (B), “(-FN)” control. Significance was determined by performing a Student’s 2-tailed t-test, assuming unequal variance between groups. (E) Jurkat (top panels) and 293T cells (bottom panels) were exposed to GFPvpr vector particles (green) for 1 hour at 37C, washed, and fixed with 4% paraformaldehyde. Cells were stained with phalloidin (red) and anti-VSV-G antibody (magenta), followed by staining with anti-rabbit Alexa Fluor 647 (blue), and imaging via deconvolution microscopy. (F) Particles either counted as “individual” (gray bars) or as “aggregate” (black bars) if 2 or more individual particles were observed to occupy contiguous space. Significance was determined by performing a Student’s 2-tailed t-test, assuming unequal variance between groups.
Figure 3.
Neutralization kinetics of cell-free vector.
(A) Vector particles were incubated in escalating concentrations of anti-VSV-G neutralizing antibody for 1 hour at 4°C in the presence of 4 µg/ml protamine sulfate. Particles were then placed on 293T cells for overnight transduction at 37°C. Cells were washed after 24 hours, and FACS was performed 48 hours later. (B) Experiment was performed as described in (A) with the exception that mouse serum was used instead of VSV-G neutralizing antibody and the neutralizing incubation was performed at 37°C for the purpose of minimizing cell death resulting from serum incubation (CD). In the cell-bound condition (black squares), 1x105 prechilled Jurkat cells were exposed to vector (C: MOI 0.3; D: MOI 3) for 1 hour at 4°C in the presence of 4 µg/ml protamine sulfate, then placed in co-culture with 2.5x104 pre-plated 293T cells at 37°C. Cell-free samples (gray circles) were prepared in parallel, in the absence of Jurkat cells. Escalating concentrations of anti-VSV-G neutralizing antibody were added to co-cultures and incubataed overnight at 37°C. Cells were then washed, and FACS was performed 48 hours later (EF). 1x105 prechilled Jurkat cells were exposed to vector (MOI 3) for 1 hour at 4°C, then placed in co-culture with 2.5x104 pre-plated 293T cells at 37°C. At serial time points (x-axis), 0.5% anti-VSV-G neutralizing antibody (E) or 10% mouse serum (F) was added to the transduction culture. Target 293T cells were washed 24 hours after vector exposure, and FACS was performed 48 hours later. CD45-APC antibody was used to exclude primary Jurkat cells (gray circles) from secondary target 293T cells (black circles). Red line depicts % 293T GFP+ events/ % Jurkat GFP+ events.
Figure 4.
Neutralization is dependent upon cell mobility.
(A) Merged z-stack of transduced adherent cell shown alongside yz and xz planes. 6.0x104 NIH 3T3 cells were allowed to adhere to a chambered well for 3 hours. Cells were then transduced for 30 minutes at 4°C, fixed with 4% paraformaldehyde, washed with PBS, permeabilized with NET buffer, stained with Alexa Fluor phalloidin 555 (red), and DAPI (blue). (B) Merged z-stack of transduced suspension cell shown alongside yz and xz planes. 6.0x104 NIH 3T3 cells were prepared as described in (A) with the exception that cells were transduced following 3 hours of suspension culture in polystyrene tubes instead of plating in adherence to chambered wells. Once cells were fixed and stained, they were placed in chambered wells for microscopy. Scale bars in (A), (B) are 5 µm. (C) The experiment described in (A) and (B) was performed twice and the average number of particles per cell was determined by counting particles in 36 cells per cohort. The frequency of cells observed to have a specific number of particles bound to the cell surface was calculated using Microsoft Excel. (D) NIH3T3 cells were exposed to increasing concentrations of vector as in (C), cells were immediately fixed, then cDNA was synthesized, and quantitative RT-PCR was performed with primers that amplified GFP (vector) or GAPDH. The relative quantitation of GFP (i.e. vector genomes) is shown on the y-axis. (E) 293T cells were allowed to adhere to tissue culture-treated plates for at least 4 hours (“Adherent”, left), or were left in suspension (F) in polypropylene tubes (“Suspension”, right). Cells were transferred to 4C for 1 hour, followed by a 1-hour vector exposure (MOI 0, 1, 3, 10). Following vector exposure, cells were transferred to 37oC and serial concentrations of anti-VSV-G neutralizing antibody were added to the media. FACS was performed 72 hours later. (G) Vector was added to pre-plated 293T cells in serially increasing concentrations while media volume and cell number remained constant (actual MOI, black circles). Increasing numbers of 293T cells were plated on 12 well poly-L-lysine-coated plates, while keeping vector concentration and media volume constant (effective MOI, gray circles). Cells were washed after 4-hour vector exposure in the presence of 4 µg/ml protamine sulfate. FACS was performed 72 hours later. (H) The experiment was performed as described in (G) with the exception that 293T cells were kept in suspension during 3-hour transduction in polystyrene tubes. Cells were then washed and transferred to 12-well plates. FACS was performed 72 hours later.
Figure 5.
Vector particles do not consistently polarize to areas of cell-cell contact.
(A) Jurkat cells were exposed to mCherryvpr vector (red) for 1 hour at 4°C, cells were washed and placed in co-culture with GFP-expressing 293T cells (green) that were pre-plated on glass cover slips. Cells were washed with PBS, stained with anti-VSV-G antibody (shown) or neutralizing anti-VSV-G antibody (not shown), fixed with 4% paraformaldehyde, and then stained with Alexa Fluor 647 anti-rabbit secondary antibody (magenta). The percentage of single or aggregated particles was enumerated in cells treated with neutralizing versus labeling VSV-G antibody. Statistical significance was determined via a 2-tailed Students t test assuming unequal variance. (B) A representative image was acquired as described in (A) and used to generate a projection image of 13 z-stacks in softWoRx Explorer. (C) 1x106 Jurkat cells were transduced overnight at 37°C (MOI 25). Cells were washed twice in the morning, pelleted, fixed for 1 hour in Karnovsky’s fixative, and prepared for electron microscopy. Panel on left is magnified 14,000x; the right top panel is magnified 36,000x. Red arrowheads highlight vector particles.
Figure 6.
Lentivector cell-cell transmission.
(A) Cell-free transduction. Several factors are shown that negatively impact transduction efficiency. (B) Cell-bound transduction. Several factors are shown that improve transduction efficiency.