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\title{Non-Viral Gene Delivery to T Cells with Lipofectamine LTX}



\author[1]{Emily Harris}%
\author[1]{Devon Zimmerman}%
\author[1]{Eric Warga}%
\author[1]{Anil Bamezai}%
\author[1]{Jacob Elmer}%
\affil[1]{Villanova University}%


\vspace{-1em}



  
  \date{June 07, 2024}


\begingroup
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\selectlanguage{english}
\begin{abstract}
Retroviral gene delivery is widely used in T cell therapies for
hematological cancers. However, viral vectors are expensive to
manufacture, they integrate genes in semi-random patterns, and their
transduction efficiency is highly variable. In this study, several
non-viral gene delivery vehicles, promoters, and additional variables
were compared to optimize non-viral transgene delivery and expression in
both Jurkat and primary T cells. Overall, transfecting Jurkat cells in
X-VIVOTM 15 media with Lipofectamine LTX provided a high transfection
efficiency (63.0\selectlanguage{ngerman}±10.9\% EGFP+). However, the same method yielded a much
lower transfection efficiency in primary T cells (8.1±0.8\% EGFP+).
Subsequent confocal microscopy revealed that a majority of the
lipoplexes did not enter the primary T cells, which might be due to
relatively low expression levels of heparan sulfate proteoglycans
(HSPGs) detected via mRNA-sequencing. PYHIN DNA sensors (e.g., AIM2,
IFI16) were also expressed at high levels in Primary T cells, which can
induce apoptosis when bound to cytoplasmic DNA. Therefore, transfection
of primary T cells appears to be limited at the level of cellular uptake
and/or DNA sensing in the cytoplasm, so both of these factors should be
considered in the development of future viral and non-viral T cell gene
delivery methods.%
\end{abstract}\selectlanguage{ngerman}%



\sloppy


\emph{}

\emph{Introduction}

Chimeric antigen receptor (CAR) T cell therapy is a type of
immunotherapy in which a patient's T cells are harvested, genetically
modified to express a chimeric antigen receptor, and then infused back
into the patient to seek out and eliminate any cells that express a
target antigen that is bound by the CAR. For example, anti-CD19 CAR-T
cell therapy is approved by the FDA for the treatment of B cell leukemia
(e.g., non-Hodgkin's lymphoma,\textsuperscript{1} chronic lymphocytic
leukemia,\textsuperscript{2} and acute lymphoblastic
leukemia\textsuperscript{3--5}). In these treatments, the CAR is
delivered to the T cells using a gammaretrovirus or
lentivirus.\textsuperscript{6,7} These viruses are generally regarded
for their high transduction efficiency, but it is worth noting that
their genomic integration patterns are
semi-random.\textsuperscript{11}For example, gammaretroviral vectors
have been shown to have a preference for integrating near
transcriptional start sites. This type of semi-random integration could
potentially lead to mutagenesis, but years of clinical scrutiny have
demonstrated these vectors to be safe thus
far.\textsuperscript{8--10,12,13} Nonetheless, these vectors are still
considered by the FDA to be potentially oncogenic and thus must be
tested for replication competence during manufacturing and patients must
be monitored for up to 15 years after receiving
treatment.\textsuperscript{13--15} It is also important to note that the
transduction efficiency achieved with retroviral delivery systems can
vary significantly between patients
(2.3-80\%),\textsuperscript{1--6}although this variation may be due to
differences between their genotypes and treatment regimens.

These challenges have motivated researchers to investigate non-viral
transfection methods for delivery of the CAR gene, which has been a
formidable task. Indeed, T cells have proven to be notoriously hard to
transfect, perhaps because they are uniquely adapted to clear the body
of viral infections and restrict viral
replication.\textsuperscript{20}For example, lymphocytes have been shown
to expel mitochondrial DNA in inflammatory webs after recognizing CpG
oligodeoxynucleotides (a pathogenic signature unique to
bacteria).\textsuperscript{7}Nonetheless, several groups have shown that
transfection efficiencies as high as 60-70\% can be achieved in primary
T cells with electroporation.\textsuperscript{21,22} This technique
applies an electric field to a sample of cells that exceeds the
capacitance of the cell membrane to create pores in the cell membrane
that allow delivery of DNA.\textsuperscript{15,23} This physical method
of introducing DNA to T cells is generally quick and inexpensive, but it
can be difficult to scale up and is relatively harsh, leading to
significant decreases in T cell viability.\textsuperscript{24--27}
Alternatively, a few groups have investigated the use of cationic
polymers (e.g., PEI and pDMAEMA) for gene delivery to T
cells.\textsuperscript{82-85} These studies have demonstrated highly
efficient delivery of siRNA and mRNA to T cells, but the maximum
transfection efficiencies for pDNA with these vehicles (1.5-25\%) tend
to be relatively low compared to electroporation and lentiviral
transduction.\textsuperscript{86}

The goal of this work was to investigate the use of Lipofectamine LTX as
a lipid-based alternative to cationic polymers, electroporation, and
lentiviral transduction for T cell gene delivery. Transfection with the
cationic lipid Lipofectamine (i.e., Lipofection) involves the formation
of a lipoplex consisting of negatively charged plasmid DNA and
positively charged liposomes. The lipoplex can then enter the cell via
endocytosis and escape into the cytoplasm by lysing the endosome through
the proton-sponge effect.\textsuperscript{28,29} The effects of
additional transfection variables (e.g., media type, promoter, et al.)
on Lipofectamine transfection efficiency were also investigated to
determine the most efficient means of non-viral gene delivery to both
Jurkat (a T cell leukemia line) and primary T cells. Finally, the
transcriptome of the T cells was also analyzed to detect potential
mechanisms of resistance to transfection or transduction.

\emph{Materials and Methods}

\emph{Plasmid/Promoter Cloning}

Initial screening experiments for the comparison of different vehicles
in Jurkat cells used either the luciferase (luc2
gene)\textsuperscript{30} expression plasmid pGL4.50 (Promega, \#E1310)
or the EGFP expression plasmid pEF-GFP (Addgene, \#11154). The pEF-Luc
plasmid used in Figures 1 \& 2 was prepared by replacing the EGFP gene
in pEF-GFP with the luc2 gene. For the promoter comparison experiments
in Figure 2, the EF1\selectlanguage{greek}α \selectlanguage{english}promoter in pEF-Luc was replaced with the CAG
promoter (cloned from Addgene plasmid \#48138) and the CMV promoter
(cloned from pGL4.50).

\emph{Jurkat T Cell Growth and Transfection}

Human T lymphocyte leukemia cells (Jurkat, clone E6-1, ATCC\selectlanguage{ngerman}®
TIB-152\textsuperscript{TM}) were seeded in 24 well plates at a density
of 2x10\textsuperscript{5} cells/well in fetal bovine serum-containing
RPMI-1640 media immediately prior to transfection. Polymer-mediated
transfections were performed by mixing either branched PEI (MW = 25
kDa), linear PEI (MW = 10 kDa), jetPEI®, or
TurboFect\textsuperscript{TM} with plasmid DNA (1,000 ng DNA/well) and
then incubating the mixture for 20 minutes at room temperature to form
polyplexes. Transfections with Lipofectamine® LTX were performed
according to the manufacturer's protocol. Branched PEI (BP), linear PEI
(LP), and jetPEI (JP) were all tested with 5:1 and 10:1
nitrogen:phosphate (N:P) ratios. N:P ratios could not be calculated for
TurboFect and Lipofectamine LTX because their formulations are
proprietary, so the following manufacturer-recommended volumes of each
vehicle were tested for the delivery of 1 \selectlanguage{greek}µ\selectlanguage{english}g pDNA/well in a 24-well
plate: ``LipoHi'' = 2.75 \selectlanguage{greek}µ\selectlanguage{english}L/well of Lipofectamine\selectlanguage{ngerman}® LTX, ``LipoLo'' =
1.25 \selectlanguage{greek}µ\selectlanguage{english}L/well of Lipofectamine\selectlanguage{ngerman}® LTX, and Turbofect = 6 \selectlanguage{greek}µ\selectlanguage{english}L/well of
TurboFect\textsuperscript{TM}. Polyplexes and lipoplexes were then added
to the cells, which were subsequently incubated for 48 hours at 37\selectlanguage{ngerman}°C.
Cells were then either analyzed for EGFP expression via flow cytometry
or lysed with 150 \selectlanguage{greek}µ\selectlanguage{english}L of cell culture lysis reagent/well (125 mM Tris-HCl
pH 7.8, 10 mM DTT, 10 mM EDTA, 50\% glycerol, and 5\% Trition X-100) to
quantify luciferase expression with a Promega Luciferase Assay Kit
(\#E1501).

\emph{Primary T Cell Growth and Transfection}

Primary T cells from three healthy donors were purchased from Astarte
Biologics and grown in X-VIVO 15 media with Human T Activator CD3/CD28
Dynabeads (Thermo Fisher Scientific \#11131D) at a 1:1 ratio of beads to
cells. The culture media was also supplemented with IL-2 at 0.5 ng/mL to
stimulate T cell growth. Beads were replaced every 7-10 days during
passaging, while IL-2 was added every 2-3 days. Transfections of primary
T cells followed the protocol that was optimized for the Jurkat T cells
(2.75 \selectlanguage{greek}µ\selectlanguage{english}L Lipofectamine and 1 \selectlanguage{greek}µ\selectlanguage{english}g of pDNA per well in a 24-well plate).

\emph{MTT Assay}

Following transfection and a 48 hour incubation period, 10 \selectlanguage{greek}µ\selectlanguage{english}L of 5 mg/mL
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)
solution was added to each well of cells and incubated for 4 hours at
37\selectlanguage{ngerman}°C to allow proliferating cells to metabolize MTT into purple
formazan. After MTT incubation, 100 \selectlanguage{greek}µ\selectlanguage{english}L of detergent (10\% SDS in 0.01\%
HCl/water) was then added to the wells and incubated for another 2 hours
at 37\selectlanguage{ngerman}°C to release and solubilize the formazan. A sample of 150 \selectlanguage{greek}µ\selectlanguage{english}L from
each well was then transferred to a clear 96-well plate and the
absorbance at 570 nm was recorded. Raw absorbance values at 570 nm for
each sample were then normalized to a non-transfected drug-free control
sample to obtain a relative measure of cell metabolic activity.

\emph{Annexin V/Propidium Iodide Cell Viability Assay}

Samples of approximately 100,000 cells were spun down at 400 g for 4
minutes, then aspirated and resuspended in BioLegend Cell Staining
Buffer two times before finally pelleting and resuspending the cells in
20 \selectlanguage{greek}µ\selectlanguage{english}L of BioLegend Annexin V Binding Buffer. 1 \selectlanguage{greek}µ\selectlanguage{english}L of propidium iodide
(Ex/Em = 493/636 nm) and 0.5 \selectlanguage{greek}µ\selectlanguage{english}L of annexin V R-PE (Ex/Em = 565/578 nm)
(Biotium \#29045) were added to each cell sample and incubated in the
dark for 20 minutes. Binding buffer (80 \selectlanguage{greek}µ\selectlanguage{english}L) was then added to each
sample and then samples were transferred to a 96-well plate for flow
cytometry.

\emph{Flow Cytometry Analysis}

Cells transfected with pEF-EGFP were analyzed 24 or 48 hours following
transfection to determine the percentage of transfected cells
(\%EGFP\textsuperscript{+}) and EGFP expression levels (mean EGFP) using
either a FACSCalibur Flow Cytometer (Figure 1) or a Millipore Sigma
Guava easyCyte HT sampling Flow Cytometer (Figures 2-6). Cells were
analyzed via side scatter analysis to differentiate live cell
populations from cell debris, while gates for fluorescent
(EGFP\textsuperscript{+}) populations were defined using non-transfected
(EGFP\textsuperscript{-}) control samples.

\emph{mRNA Sequencing}

To detect potential changes in host cell gene expression patterns after
transfection, parallel cultures of PC-3, Jurkat, and primary T cells
were passaged into fresh plates or flasks. Control (untransfected)
cultures were left to grow for 48 hours, while the remaining cultures
were transfected with pEF-GFP one day after passaging. On the following
day, a small fraction (10\%) of each culture was then analyzed using
flow cytometry and fluorescent microscopy to confirm EGFP expression,
then total RNA samples were extracted from the remaining cells with a
Qiagen RNEasy kit. The RNA samples were then submitted to the Beijing
Genomics Institute (BGI, Hong Kong, China) for library preparation,
next-generation sequencing, and data analysis to obtain the FPKM values
shown in Tables 1-3.

\emph{}

\emph{Fluorescent Plasmid Transfections}

Transfections of Mirus Bio Label IT fluorescein-labeled plasmid (MIR
7906) were conducted by mixing 2.75 \selectlanguage{greek}µ\selectlanguage{english}L Lipofectamine with 1 \selectlanguage{greek}µ\selectlanguage{english}g pDNA for
each well of 2x10\textsuperscript{5} Jurkats and
CD3\textsuperscript{+}primary T cells, while wells with
1x10\textsuperscript{5} PC-3 cells were transfected with 1 \selectlanguage{greek}µ\selectlanguage{english}L Lipo and
500 ng pDNA. Jurkat and primary T cells were both cultured and
transfected in serum-free X-VIVO 15 media, while PC-3 cells were
cultured and transfected in RPMI 1640 media supplemented with 10\% FBS.
Following transfection, all cells were incubated for 24 hours and then
spun down at 300 g for 4 minutes to remove excess pDNA in the media. The
cells were then resuspended in trypsin-EDTA solution and incubated for
10, 20, or 30 minutes to disrupt interactions between cell surface
proteins and lipoplexes. Once the incubation was complete, the trypsin
was quenched with serum, the cells were spun down again, resuspended in
phosphate buffered saline (PBS) that was supplemented with 10\% FBS, and
then analyzed for fluorescein labeling via flow cytometry. The same
protocol was followed for the PC-3 cells, but with the addition of an
initial trypsinization to detach the cells from the culture plate.

\emph{Confocal Microscopy Slide Preparation}

Slides were prepared for confocal microscopy with a standard fixing,
washing, staining, and mounting protocol.\textsuperscript{9} PC-3 cells
were grown for 48 hours on \#1.5 glass coverslips after transfection and
then excess media was removed before fixing for 10 minutes in 4\%
formaldehyde solution, followed by three washes with 1X PBS. Biotium Red
CellBrite Cytoplasmic Membrane dye was diluted according to the
manufacturer's protocol (5 \selectlanguage{greek}µ\selectlanguage{english}L of cell staining solution in 1 mL of PBS)
and then added to the coverslips, which were incubated for 10 minutes in
the dark and then washed three more times. Two drops of Thermo Fisher
NucBlue Live ReadyProbes Reagent (Hoescht 33342) were then added to
the slips in 1 mL of PBS and incubated in the dark for 20 minutes.
Finally, 10 \selectlanguage{greek}µ\selectlanguage{english}L of 1X PBS was used as mounting medium to mount the
coverslips onto AmScope microscope slides (BS-72P) and sealed with clear
nail polish. The same protocol was followed for primary T cells with 300
g for 4 minute centrifugation spins in between each step.

All images were obtained using a Leica TCS SP8 Four Channel Confocal
Microscope with a 63X oil immersion lens. Lasers were set for 407 nm
(DAPI), 488 nm (EGFP), and 633 nm (AlexaFluor 647). A three-sequence run
was used to maximize reading while minimizing background and crosstalk.
DAPI was set to PMT while EGFP and Alexa Fluor 647 were set to HyD.
Images were taken using z-stacking from the bottom to the top of each
cell.

\emph{Statistical Analysis}

\emph{Results and Discussion}

\emph{Non-Viral Gene Delivery Vehicles}

An initial screen of several cationic polymers (branched PEI, linear
PEI, jetPEI, \& Turbofect) and lipids (Lipofectamine LTX) in Jurkat T
cells revealed that the vehicles could be separated into three
significantly different cohorts (Figure 1A). For example, branched PEI
and some of the other polymeric vehicle formulations provided no
detectable luciferase expression, while Turbofect and a specific
nitrogen:phosphate ratio (N:P = 10:1) of linear PEI yielded modest
luciferase expression. The highest luciferase expression levels were
obtained with Lipofectamine LTX (Lipo-Lo = 1.25 \selectlanguage{greek}µ\selectlanguage{english}L and 1\selectlanguage{greek}μ\selectlanguage{english}g DNA; Lipo-Hi
= 2.75 \selectlanguage{greek}µ\selectlanguage{english}L and 1 \selectlanguage{greek}μ\selectlanguage{english}g DNA) and jetPEI (N:P = 5:1).

Subsequent transfections of pEF-GFP (Figure 1B) using the vehicles that
provided the highest luciferase expression levels (Lipofectamine,
Turbofect, and jetPEI) showed that the higher dose of Lipofectamine LTX
(Lipo-Hi) provided the highest transfection efficiency for Jurkat T
cells (25\% EGFP\textsuperscript{+}), while the transfection
efficiencies obtained with Turbofect and jetPEI were significantly lower
(4.5-4.8\% EGFP\textsuperscript{+} cells). As observed with the
luciferase assays, the higher amount of Lipofectamine (Lipo-Hi) appeared
to provide a higher number of EGFP\textsuperscript{+} cells than the
lower amount (Lipo-Lo), but the difference was not statistically
significant. The histograms in Figure 1D-F also illustrate a distinct
increase in the number of fluorescent cells obtained with Lipo-Hi
compared to Lipo-Lo and Turbofect.

The effects of Lipofectamine, Turbofect, and jetPEI (5:1) on cell
metabolic activity were also measured with an MTT assay (Figure 1C).
Each vehicle significantly decreased cell metabolic activity
approximately 30-40\% relative to control samples of untransfected
cells, but no significant differences were observed between the vehicles
themselves.

Overall, Lipofectamine LTX appeared to be more efficient than Turbofect
and branched or linear PEI. This observation is in agreement with
previous studies that have compared the transfection efficiencies of
cationic lipids and polymers in other cell lines.\textsuperscript{31,32}
These studies attribute the higher transfection efficiency of the lipid
vehicles to their higher affinity for cell membranes, which allows them
to be endocytosed more quickly than cationic polymers like
PEI.\textsuperscript{31}Consequently, Lipofectamine (Lipo-Hi) was
selected as the vehicle for our subsequent studies.

\emph{Promoters and Plasmids}

While cationic polymers and lipids play an important role in delivering
genes to cells, promoters play an equally important role in ensuring the
expression of the gene that must not be overlooked. Indeed, transgene
expression has been shown to vary significantly for different promoters
between cell types.\textsuperscript{33,34} For example, the mammalian
EF1\selectlanguage{greek}α \selectlanguage{english}promoter provides high levels of transgene expression in mouse
embryonic cell lines, while expression from the viral cytomegalovirus
(CMV) promoter is significantly lower in those
cells.\textsuperscript{35}

Figure 2 shows a comparison of three commonly used promoters (EF1\selectlanguage{greek}α, \selectlanguage{english}CMV,
and the CMV early enhancer/chicken beta-actin/rabbit beta globin hybrid
promoter known as ``CAG'') that were used to drive expression of
luciferase (Figure 2A) and EGFP (Figure S1) in Jurkat T cells. Similar
trends in luciferase expression were observed with both Lipofectamine
and jetPEI, with the EF1\selectlanguage{greek}α \selectlanguage{english}promoter showing the highest luciferase levels
followed by a slightly lower (but not statistically significant)
decrease in the level of luciferase expression provided by the CAG
promoter. A significantly lower level of luciferase expression was
observed with the viral CMV promoter, which was the only promoter in
these experiments that lacked an intron. This may explain the decrease
in luciferase expression levels, since introns have been previously
shown to increase transgene expression in other cell
types.\textsuperscript{36} However, it is also interesting to note that
no significant difference in GFP expression was observed between the
promoters (Figure S1).

\emph{Effects of Cell Culture Media}

In addition to vehicles and promoters, the effects of several other
variables were also tested to optimize transfection efficiency in Jurkat
T cells. For example, cells were treated with small molecule inhibitors
that have previously been shown to enhance transgene expression in other
cell types (e.g., BX795, MS-275, AG-490, HMN-214 et
al).\textsuperscript{37,38} One of the inhibitors (iCRT14) enhanced
luciferase expression approximately 3-fold, but did not significantly
enhance transfection efficiency (i.e., \%EGFP\textsuperscript{+} cells,
shown in Figure S2). We also compared the conventional pEF-GFP plasmid
to EGFP-expressing nanoplasmids (Nature Technologies, \#NTC9385R), which
are much smaller (1,594 bp) than pEF-EGFP (5,051 bp) and have been shown
to increase transfection efficiency and the duration of EGFP expression
in other cell lines and \emph{in vivo} .\textsuperscript{39,40} However,
at both 24 and 48 hour time points following transfection, no
significant differences in EGFP expression were observed between pEF-GFP
and the nanoplasmids in Jurkats. Finally, varying amounts of
Lipofectamine did not have a significant effect on transfection
efficiency was observed (Figure 3A), but the lowest dose of
Lipofectamine was significantly less toxic to the Jurkat T cells (Figure
3B).

One variable that did significantly influence transfection efficiency
was the type of media used during the transfection. For example, the
initial experiments shown in Figures 1 and 2 were conducted in
serum-containing RPMI-1640 media, since that is the media recommended by
ATCC for Jurkat cells (clone E6-1, TIB-152). In contrast,
chemically-defined serum-free media formulations like X-VIVO 15 are
typically used in primary T cell culture to avoid the potentially
problematic effects of serum components \emph{in vivo} . As shown in
Figure 3C, culturing Jurkat cells in X-VIVO media significantly
increased the transfection efficiency of Lipofectamine in Jurkat cells
(63.0\selectlanguage{ngerman}±10.9\% EGFP\textsuperscript{+}), relative to the modest
transfection efficiency observed in RPMI media (23.1±5.5\%
EGFP\textsuperscript{+}). Similar results were also observed with
transfections of nanoplasmids (Figure S3).

Since albumin and other components present in serum-containing (SCM)
RPMI are known to inhibit transgene
delivery,\textsuperscript{41,42}additional transfections were performed
with serum-free (SFM) RPMI and serum-containing X-VIVO media. The
exclusion of serum from RPMI had no significant effect on transfection
efficiency, but the addition of serum to the X-VIVO media did
significantly decrease transfection efficiency. However, the
transfection efficiency obtained with serum-containing X-VIVO media was
still higher than both SCM- and SFM-RPMI at 24 and 48 hours
post-transfection, which suggests that some component of the X-VIVO
media may enhance transfection. For example, one component that is
present in X-VIVO but absent in RPMI is recombinant transferrin, which
has previously been shown to increase Lipofection efficiency by
enhancing endocytosis and nuclear targeting.\textsuperscript{43,44}

\emph{Lipofection of Primary T Cells}

After determining the optimum vehicle (Lipofectamine LTX), promoter
(EF1\selectlanguage{greek}α\selectlanguage{english}), and media formulation (serum-free X-VIVO) in Jurkat cells, we
used the same conditions to transfect CD3\textsuperscript{+} primary T
cells. Surprisingly, this protocol yielded much lower transfection
efficiencies in the primary T cells (8.1\selectlanguage{ngerman}±0.8\% EGFP\textsuperscript{+})
than the Jurkat T cells (Figure 4A). This dramatic decrease in transgene
expression is also illustrated by the histograms in Figures 4C/D and was
visually apparent when observing the cells with fluorescent microscopy,
since the Jurkat cells fluoresced brightly while the primary T cells
were relatively dim. It is worth noting that primary T cells are
cultured with additional components (recombinant IL-2 and anti-CD3/CD28
Dynabeads) that could potentially interfere with transfection, but
culturing Jurkat T cells with Dynabeads and IL-2 did not significantly
decrease transfection efficiency (data not shown).

A previous study using cationic pHEMA-g-pDMAEMA polymers observed a
similar disparity, in which the polymer provided 50\% transfection
efficiency in Jurkat T cells but only 18-25\% transfection efficiency in
CD4\textsuperscript{+} and CD8\textsuperscript{+} primary T
cells.\textsuperscript{45} This same group later showed that endosomes
in primary T cells acidify at a slower rate than endosomes in HeLa
cells, which could slow the rate of endosomal escape for non-viral gene
delivery vehicles that rely upon the proton sponge effect to destabilize
the endosome and escape into the cytoplasm.\textsuperscript{73}

Another variable that significantly influence transfection efficiency in
primary T cells was the timing (0-3 days) between activation and
transfection (Figure 4B). Indeed, other groups have previously shown
that the time between these two steps significantly impacts gene
delivery with viruses, electroporation, and cationic
polymers.\textsuperscript{25,45,12--14} In our experiments, cells were
either transfected 30 minutes after the addition of anti-CD3/28
Dynabeads to the cells (i.e., Day 0) or 24, 48, and 72 hours later
(i.e., Days 1-3). Interestingly, transfection efficiency was highest
when lipoplexes were added to the cells on Day 0 or Day 1, but decreased
significantly on Days 2 and 3. This downward trend in transfection may
be due to the fact that cell size and substrate uptake are both
maximized immediately after
activation.\textsuperscript{74,75}Additionally, cell division spikes
after activation, which would allow for more effective entry into the
nucleus compared to cells that are in a resting
state.\textsuperscript{76}

\emph{Tracking Plasmid Delivery Using a Fluorescent Plasmid in T cells}

The relatively low transfection efficiency that was obtained with
Lipofectamine in the primary T cells could be due to multiple different
steps in the transfection process, including cellular uptake, nuclear
translocation, transcription, and translation of the transgene. However,
since other groups have achieved high transfection efficiencies (up to
70\%) with electroporation of similar plasmids and synthetic mRNAs in
primary T cells,\textsuperscript{24,46--48} we hypothesized that
cellular uptake may be the key limiting step for Lipofectamine (instead
of nuclear uptake or transcription/translation). To test this
hypothesis, we used a fluorescein-labeled plasmid (Mirus Bio\selectlanguage{english}, MIR 7906)
to track the delivery of fluorescent plasmid DNA to the cells with flow
cytometry and confocal microscopy.

First of all, as shown in Figure 5A, transfection of adherent PC-3 cells
(a cell line that is relatively permissive to transfection) with
Lipofectamine and pEF-GFP provided a very high percentage of
EGFP\textsuperscript{+} cells (91+-1.1\% EGFP\textsuperscript{+}).
Likewise, a similarly high percentage of the PC-3 cells fluoresced when
transfected with the fluorescein-labeled plasmid (96+-0.94\%
fluorescein\textsuperscript{+}), demonstrating that the plasmid was
successfully delivered to almost all the PC-3 cells. Interestingly, the
transfection efficiency observed with pEF-GFP for the Jurkat cells
(51+-9.7\% EGFP\textsuperscript{+}) was more modest, but the percentage
of fluorescein+ cells (74+-4.5\%) was significantly higher. The mean
fluorescence (Figure 5B) of the PC-3 cells was also significantly higher
than the mean fluorescence of the Jurkat cells in both types of
transfections.

Similar trends were also observed with the primary T cells (Figure
5A/B). The percentage of fluorescein\textsuperscript{+} primary T cells
and the mean fluorescence of the fluorescein\textsuperscript{+} primary
T cells was significantly lower than both Jurkat T cells and the PC-3
cells. There was also a more dramatic disparity between the fraction of
fluorescein\textsuperscript{+} primary T cells (51+-11.2\%
EGFP\textsuperscript{+}) and the percentage of primary T cells
expressing EGFP (8.1+-0.8\% EGFP\textsuperscript{+}). This stark
difference suggests that the lipoplexes bind to a large fraction of the
primary T cells, but many of those lipoplexes may fail to either enter
the cell via endocytosis, escape the endosome into the cytosol, or enter
the nucleus.

To determine if the fluorescent lipoplexes were bound to the surface of
the cells or taken up into the cytoplasm or nucleus, cells transfected
with the fluorescein-labeled plasmid were treated with trypsin-EDTA for
up to 30 minutes to disrupt any potential interactions between cell
surface proteins on the cell surface and the lipoplex (Figure 5C/D).
Hypothetically, if the fluorescent lipoplexes were simply bound to the
cell membrane, then this trypsinization would detach them, leading to a
significantly lower mean fluorescence and percentage of
fluorescein\textsuperscript{+} cells. Indeed, trypsinization had no
significant effect on the percentage of
fluorescein\textsuperscript{+}PC-3 cells or their mean fluorescence,
suggesting that most of the fluorescent lipoplexes were in the cytosol
or nucleus of the PC-3 cells. In contrast, trypsinization significantly
decreased the mean fluorescence and the percentage of
fluorescein\textsuperscript{+} Jurkat cells to a level that was
comparable to the percentage of EGFP\textsuperscript{+} cells shown in
Figure 5A. A similar, although not statistically significant, decrease
in the percentage of fluorescein\textsuperscript{+} cells was also
observed in the primary T cells. However, the mean fluorescence of the
primary T cells significantly decreased and was virtually eliminated
after 30 minutes of trypsinization (Figure 5D). Altogether, these
findings suggest that cellular uptake may be a limiting step for gene
delivery in both Jurkat and primary T cells.

To further investigate the localization of the fluorescein-labeled
plasmid in the PC-3 and primary T cells, fluorescent microscopy was used
to visualize the fluorescent lipoplexes after transfection (Figure 6).
In these experiments, cells were also stained with Hoescht 33342 nuclear
stain and the Biotium CellBrite red cytoplasmic membrane dye to
visualize the nucleus and cytoplasm, respectively. Overall, similar
trends in fluorescence were observed across the cell lines. A widespread
distribution of bright EGFP fluorescence within the cytoplasm was
observed in PC-3 cells transfected with pEF-EGFP (indicating successful
transgene expression), while cells transfected with the
fluorescein-labeled plasmid exhibited small regions of concentrated
fluorescence inside the cytoplasm and nucleus that indicated successful
plasmid uptake and nuclear delivery (Figure 6A). In addition, z-stacking
with 0.5 \selectlanguage{greek}µ\selectlanguage{english}m slices from the top edge to the bottom edge of the PC-3
cells also confirmed that the fluorescent lipoplexes were inside the
cell (individual images areshown in Figure 6C, while compiled z-stacks
are shown in a video in Figure S5).

In contrast, transfection of CD3\textsuperscript{+} primary T cells with
pEF-EGFP only yielded a small fraction (3.9\selectlanguage{ngerman}±0.24\%) of
EGFP\textsuperscript{+} cells and the primary T cells that did fluoresce
were much dimmer than the PC-3 cells (in agreement with the flow
cytometry data shown in Figure 5B). Confocal microscopy z-stacking
images also showed that the fluorescent lipoplexes appeared to be
localized to the outside of the cell membrane instead of being
internalized by the cell (Figures 6C and S6). These observations further
support the notion that lipoplexes may successfully bind to primary T
cells, but endocytosis of the lipoplexes appears to be limited.

\emph{Transcriptome Analysis of Primary T Cells}

The experiments with the fluorescein-labeled plasmid (Figures 5-6)
seemed to suggest that impaired cellular uptake of the plasmid may be
partially responsible for the T cells' resistance to transfection, but
the observation that 30-40\% of the primary T cells remained
fluorescein\textsuperscript{+} after trypsinization suggested that other
intracellular mechanisms may also impair endosomal escape, nuclear
delivery, and/or expression of the transgene. In an effort to detect any
additional mechanisms that could inhibit transgene delivery or
expression and provide a rationale for the relatively low transfection
efficiency of primary T cells, mRNA sequencing was used to investigate
the transcriptomes of PC-3, Jurkat, and primary T cells in the absence
or presence of lipoplexes. These experiments were motivated by previous
studies that showed both viral vectors and Lipofectamine can activate
innate immune response pathways which trigger the expression of
anti-viral genes (TLRs, MyD88, IRFs) that can hinder transduction in
some cell types.\textsuperscript{54}

The complete mRNA-seq data (fastq and bam files, along with a
spreadsheet of all FPKM values) from these experiments are available at
the NCBI GEO repository (GEO Accession\# GSE151759). Overall, one of the
most significant differences that was observed in the gene expression
profiles of the PC-3 cells and both types of T cells (Table 1) was the
absence of several heparan sulfate proteoglycans (HSPGs), which are also
known as syndecans (SDCs). As shown in Table 1, HSPG2 and all the
sydecans were expressed in PC-3 cells, but mostly absent in both Jurkat
and primary T cells (with and without lipoplexes). SDC3 was the only
syndecan expressed in Jurkat cells, but its role in endocytosis and gene
delivery has not yet been established. SDC4 was expressed in primary T
cells (as expected for activated T cells), but SDC2 was not detected.
Similar results were reported by one study that showed 100-fold lower
levels of HSPG expression in Jurkat T cells compared to HeLa
cells.\textsuperscript{77} Low levels of HSPG expression have also been
observed in primary T cells, but T cell activation can upregulate SDC2
and SDC4 expression.\textsuperscript{58,78}

This general lack of syndecan expression may explain the significantly
lower transfection efficiencies shown in Figure 5A for the Jurkat and
primary T cells relative to the PC-3 cells (which express HSPG2 and all
the syndecans). Indeed, while HSPGs are best known for their roles in
the attachment of adherent cells to the extracellular matrix or tissue
culture plates, they are also directly involved in gene delivery, since
they regulate endocytosis and their negatively charged sulfate groups
are involved in the initial binding of several viruses and positively
charged polyplexes or lipoplexes.\textsuperscript{79} Indeed,
overexpression of SDC1, SDC2, and SDC4 enhances the transfection
efficiency of liposomes in K562 cells, although overexpression of SDC2
has also been shown to inhibit PEI-mediated gene
delivery.\textsuperscript{67,80} Alternatively, blocking the sulfation
of SDCs has also been shown to inhibit endocytosis and gene
delivery.\textsuperscript{57,67}

As previously mentioned, mRNA-sequencing was performed in the different
cell lines both in the absence and presence of lipoplexes. The rationale
for comparing transfected and untransfected cells was to determine if
there were any host cell genes that were upregulated in response to
transfection of double-stranded plasmid DNA. Indeed, there are many
examples of genes that are induced or upregulated when dsDNA is detected
in the cytoplasm and many of these upregulated genes have potent
anti-viral functions that are designed to inhibit the replication of
viruses.\textsuperscript{68,69} Unfortunately, many of these genes can
also inhibit non-viral transgene delivery or
expression.\textsuperscript{70}

Transfection of PC-3 cells led to the induction or upregulation of
hundreds of cytokines and cytokine-stimulated genes (CSGs, data not
shown), some of which can inhibit transgene delivery (e.g., IFITM 1, 2,
and 3) or translation (e.g., IFIT 1 and 2). Nonetheless, the
transfection efficiency and mean GFP levels were still high in PC-3
cells, suggesting that many of these upregulated genes may be
inconsequential for non-viral pDNA or transgene delivery and expression
in PC-3 cells.

In contrast to PC-3 cells, only a few differentially expressed genes
were significantly upregulated at least 3-fold in the Jurkat and primary
T cells (Table 2). This result is somewhat unanticipated, since the
requisite cytosolic DNA sensors (e.g., cGAS and IFI16) and the
downstream effectors (e.g., STING, TBK1, and IRFs) that are necessary to
detect foreign DNA and induce the expression of cytokines and CSGs were
detected in the Jurkat and primary T cells (data not shown). This lack
of an innate immune response to dsDNA has been previously reported by
other groups, suggesting that T cells may lack an unknown component of
the DNA sensing pathways or they somehow repress
CSGs.\textsuperscript{8} Nonetheless, some metallothioneins were
significantly upregulated in the Jurkat (MT1F, MT2A) and primary T cell
lines (MT1H) after transfection. Metallothioneins are involved mainly in
metal binding, often to zinc, but they have also been implicated in
immune regulation and the response to bacterial and viral
infections.\textsuperscript{60,61} However, the role of metallothioneins
in transgene delivery and expression has not yet been determined.

Although T cells did not significantly upregulate cytokines or CSGs in
response to transfection, one interesting trend that emerged when
comparing the transcriptomes of the T cells to the PC-3 cells was that
multiple CSGs that were upregulated in PC-3 cells after transfection
were constitutively expressed at relatively high levels in the T cell
lines (Table 2). For example, each member of the pyrin and HIN (PYHIN)
domain DNA sensor family (IFI16, AIM2, and PYHIN1/IFIX) was detected in
both untransfected and transfected primary T cells. In contrast, AIM2
was only expressed in PC-3 cells after transfection and IFI16 was highly
upregulated in transfected PC-3 cells, suggesting that these DNA sensors
play an important role in the innate immune response to dsDNA in PC-3
cells. This is a particularly intriguing observation, because after AIM2
and IFI16 bind dsDNA in the cytoplasm, they form an inflammasome complex
with PYCARD, Caspase 1/8, and Gasdermin D (all of which were expressed
at detectable levels in primary T cells, but not Jurkat cells) that can
induce inflammation, apoptosis, and
pyroptosis.\textsuperscript{64}Pyroptosis has also been observed in
primary T cells during abortive HIV infection, in which double-stranded
cDNA is generated in the cytoplasm by the virus.\textsuperscript{63}
Therefore, while the AIM2 and IFI16 inflammasome pathways must be
induced (AIM2 \& CASP1) or upregulated in PC-3 cells, it appears that
primary T cells constitutively express the genes in these pathways,
which may lead to higher levels of apoptosis upon transfection with
plasmid DNA and the decrease in proliferation shown in Figure 1. Indeed,
several other studies have reported that dsDNA is highly toxic in T
cells.\textsuperscript{71,72}

\emph{Conclusions}

Overall, this study shows that Lipofectamine can be used to transfect a
moderately high percentage of Jurkat cells (51±9.7\%) but a much lower
fraction (8.1±0.8\%) of primary T cells. Nonetheless, this modest
transfection efficiency may still be suitable for the generation of
CAR-T cells, since some studies using lentiviral vectors have obtained
complete responses with transduction efficiencies as low as 2.3\%
CAR\textsuperscript{+} T cells.\textsuperscript{81} In either case, our
results emphasize that cellular uptake (due to a lack of HSPGs) and an
increased risk of apoptosis induced by the PYHIN DNA sensors AIM2 and
IFI16 are two particularly important issues that should be considered in
the development of both viral and non-viral gene delivery methods for T
cells.

\emph{Acknowledgements}

This work was supported by grants from the National Science Foundation,
USA (\#1645225 and \#1651837 to JE).

Statement of contribution of all authors: DZ and EH performed the
research, JE designed the research study, AB assisted in conducting and
analyzing the results of flow cytometry experiments, DZ and EH analyzed
the data, JE and EH wrote the paper.

\emph{Data Availability Statement}

The complete mRNA-seq data (fastq and bam files, along with a
spreadsheet of all FPKM values) from these experiments are available at
the NCBI GEO repository (GEO Accession\# GSE151759).

\emph{Conflict of Interest Declaration}

The authors have no conflicts of interest to disclose.

\emph{ORCID}

Jacob Elmer: https://orcid.org/0000-0001-6980-489X

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\emph{}

\emph{Tables}

\textbf{Table 1 -- Expression levels (FPKM) of HSPGs in PC-3, Jurkat,
and primary T cells.}\selectlanguage{english}
\begin{longtable}[]{@{}lllllll@{}}
\toprule
& \emph{Primary T cells} & \emph{Primary T cells} & \emph{Jurkat T
cells} & \emph{Jurkat T cells} & \emph{PC-3 cells} & \emph{PC-3
cells}\tabularnewline
Gene & Control & Trans. & Control & Trans. & Control &
Trans.\tabularnewline
HSPG2 & 0.1 & 0.1 & 0.1 & 0.1 & 17.7 & 14.6\tabularnewline
SDC1 & 0.1 & 0.1 & 0.1 & 0.1 & 74.0 & 31.0\tabularnewline
SDC2 & 0.0 & 0.0 & 0.1 & 0.0 & 12.8 & 6.5\tabularnewline
SDC3 & 0.0 & 0.0 & 10.0 & 11.6 & 5.4 & 3.9\tabularnewline
SDC4 & 95.3 & 103.9 & 0.0 & 0.0 & 30.0 & 44.7\tabularnewline
\bottomrule
\end{longtable}

\textbf{Table 2 -- Expression levels (FPKM) of upregulated host cell
genes in PC-3, Jurkat, and primary T cells.}\selectlanguage{english}
\begin{longtable}[]{@{}lllllll@{}}
\toprule
& \emph{Primary T cells} & \emph{Primary T cells} & \emph{Jurkat T
cells} & \emph{Jurkat T cells} & \emph{PC-3 cells} & \emph{PC-3
cells}\tabularnewline
Gene & Control & Trans. & Control & Trans. & Control &
Trans.\tabularnewline
MT1H & 0.1 & 23.8* & \emph{n.d.} & \emph{n.d.} & \emph{n.d.} &
\emph{n.d.}\tabularnewline
MT1E & 108.8 & 260.4 & 0.7 & 4.0* & \emph{n.d.} &
\emph{n.d.}\tabularnewline
MT1F & 12.4 & 46.6 & 1.0 & 3.3* & 5.8 & 40.5*\tabularnewline
\emph{n.d. = not detected, asterisks (*) indicate significant
upregulation vs. untransfected controls, values shown are the average of
triplicate (n = 3) measurements} & \emph{n.d. = not detected, asterisks
(*) indicate significant upregulation vs. untransfected controls, values
shown are the average of triplicate (n = 3) measurements} & \emph{n.d. =
not detected, asterisks (*) indicate significant upregulation vs.
untransfected controls, values shown are the average of triplicate (n =
3) measurements} & \emph{n.d. = not detected, asterisks (*) indicate
significant upregulation vs. untransfected controls, values shown are
the average of triplicate (n = 3) measurements} & \emph{n.d. = not
detected, asterisks (*) indicate significant upregulation vs.
untransfected controls, values shown are the average of triplicate (n =
3) measurements} & \emph{n.d. = not detected, asterisks (*) indicate
significant upregulation vs. untransfected controls, values shown are
the average of triplicate (n = 3) measurements} & \emph{n.d. = not
detected, asterisks (*) indicate significant upregulation vs.
untransfected controls, values shown are the average of triplicate (n =
3) measurements}\tabularnewline
\bottomrule
\end{longtable}

\textbf{Table 3 -- Expression levels of PHYIN DNA sensor pathway genes
in PC-3, Jurkat, and primary T cells}\selectlanguage{english}
\begin{longtable}[]{@{}lllllll@{}}
\toprule
& \emph{Primary T cells} & \emph{Primary T cells} & \emph{Jurkat T
cells} & \emph{Jurkat T cells} & \emph{PC-3 cells} & \emph{PC-3
cells}\tabularnewline
Gene & Control & Trans. & Control & Trans. & Control &
Trans.\tabularnewline
IFI16 & 136.1 & 109.9 & 227.4 & 212.3 & 10.9 & 407.9*\tabularnewline
AIM2 & 14.4 & 17.6 & \emph{n.d.} & \emph{n.d.} & \emph{n.d.} &
4.4*\tabularnewline
PYHIN1 & 9.9 & 5.3 & 0.6 & 0.5 & \emph{n.d.} &
\emph{n.d.}\tabularnewline
PYCARD & 33.9 & 12.6 & \emph{n.d.} & \emph{n.d.} & 2.7 &
3.1\tabularnewline
CASP1 & 15.0 & 12.3 & \emph{n.d.} & \emph{n.d.} & \emph{n.d.} &
25.0*\tabularnewline
CASP8 & 22.2 & 17.0 & 40.6 & 37.8 & 16.4 & 46.9*\tabularnewline
GSDMD & 22.8 & 19.8 & 29.6 & 31.9 & 31.1 & 106.6*\tabularnewline
\emph{n.d. = not detected, asterisks (*) indicate significant
upregulation vs. untransfected controls, values shown are the average of
triplicate (n = 3) measurements} & \emph{n.d. = not detected, asterisks
(*) indicate significant upregulation vs. untransfected controls, values
shown are the average of triplicate (n = 3) measurements} & \emph{n.d. =
not detected, asterisks (*) indicate significant upregulation vs.
untransfected controls, values shown are the average of triplicate (n =
3) measurements} & \emph{n.d. = not detected, asterisks (*) indicate
significant upregulation vs. untransfected controls, values shown are
the average of triplicate (n = 3) measurements} & \emph{n.d. = not
detected, asterisks (*) indicate significant upregulation vs.
untransfected controls, values shown are the average of triplicate (n =
3) measurements} & \emph{n.d. = not detected, asterisks (*) indicate
significant upregulation vs. untransfected controls, values shown are
the average of triplicate (n = 3) measurements} & \emph{n.d. = not
detected, asterisks (*) indicate significant upregulation vs.
untransfected controls, values shown are the average of triplicate (n =
3) measurements}\tabularnewline
\bottomrule
\end{longtable}

\emph{}

\emph{Figure Legends}

\textbf{Figure 1:} Comparison of non-viral gene delivery vehicles in
Jurkat T cells, with the lead vehicle (Lipo-Hi) represented by black
bars. Each vehicle was used to deliver either the luciferase expression
plasmid pGL4.50 (A) or the EGFP expression plasmid pEF-GFP (B-F). Panels
D-F show representative histograms (cell count vs. cell fluorescence,
FL1-H) for Turbofect and 2 concentrations of Lipofectamine. Gates used
to calculate \%EGFP\textsuperscript{+} cells are shown as horizontal
bars near the top of each panel. Panel C shows the effects of lead
vehicles from (A) on cell metabolic activity as measured with MTT as an
indication of overall cell viability. Statistically similar cohorts are
grouped with letters in (A) and (B), while an asterisk (*) indicates
significantly lower values than the control (p\textless{}0.05) in (C).

\textbf{Figure 2:} Comparison of common promoters (EF1\selectlanguage{greek}α, \selectlanguage{english}CAG, and CMV)
in Jurkat T cells. (A) Expression of luciferase (LUC) from each promoter
following delivery with either Lipofectamine (Lipo-Hi) or jetPEI (N:P =
5:1). (B) Transfection efficiencies (\%EGFP\textsuperscript{+}

\textbf{Figure 3:} ~Lipofectamine volume (\selectlanguage{greek}µ\selectlanguage{english}L) impact on transfection
efficiency and viability in Jurkat cells. (A) Transfection efficiency at
24 and 48 hours post-transfection and (B) Annexin V/PI viability
staining at 24 hours post-transfection. (C) Comparison of the effects of
media type (RPMI-1640 and X-VIVO\textsuperscript{TM}

\textbf{Figure 4:} Lipofection of Primary CD3\textsuperscript{+} T
cells. (A) Transfection efficiency of Jurkats and primary
CD3\textsuperscript{+} T cells at optimized conditions (2.75 \selectlanguage{greek}µ\selectlanguage{english}L Lipo, 1
\selectlanguage{greek}µ\selectlanguage{english}g pEF-EGFP, X-VIVO media, transfected 24 hours post-passage and 30 min
after activation). (B) Transfection efficiency of
CD3\textsuperscript{+}T cells at different days post-activation with
Dynabeads\textsuperscript{}. (C) Representative FACS histogram denoting
untransfected and transfected Jurkat T cells.

\textbf{Figure 5:} Flow cytometry analysis of PC-3, Jurkat, and
CD3\textsuperscript{+} primary T cells transfected with either pEF-GFP
or a fluorescein-labeled plasmid. (A) Percentage of fluorescent cells 24
hours transfection with either pEF-GFP or the fluorescein-labeled
plasmid (B) Mean fluorescence of transfected cells 24 hours
post-transfection. (C) Effects of trypsinization on the percentage of
fluorescein\textsuperscript{+} cells and their (D) mean fluorescence
levels. Error bars denote one standard deviation. For (A) and (B), CRD
ANOVA and Kruskal-Wallis test used to calculate significance within cell
lines. For (C) and (D), significance calculated within cell lines using
CRD ANOVA and Tukey post-hoc or Kruskal-Wallis test, across time points.

\textbf{Figure 6:} Confocal microscopy images of PC-3 and
CD3\textsuperscript{+} primary T cells transfected with pEF-EGFP and
Fluorescein fluorescent plasmid. (A) Confocal microscopy images of PC-3
cells transfected with pEF-EGFP and Fluorescein at 48 hours
post-transfection (B) Confocal microscopy images of
CD3\textsuperscript{+} primary T cells at 24 hours post-transfection.
(C) Confocal microscopy images/Z-stacks of PC-3 and Primary T cells
transfected with fluorescein-labeled pDNA and then stained with Hoescht
33342 and CellBrite Red.\selectlanguage{english}
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\end{figure}\selectlanguage{english}
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\end{center}
\end{figure}\selectlanguage{english}
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\includegraphics[width=0.70\columnwidth]{figures/Figure-3/Figure-3}

\end{center}
\end{figure}\selectlanguage{english}
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\includegraphics[width=0.70\columnwidth]{figures/Figure-4/Figure-4}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Figure-5/Figure-5}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Figure-6/Figure-6}

\end{center}
\end{figure}

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