gene transfection and expression in resting and activated murine cd4 t cell subsets
TRANSCRIPT
www.elsevier.com/locate/jim
Journal of Immunological Methods 282 (2003) 93–102
Gene transfection and expression in resting and activated
murine CD4 T cell subsets
Wendy Laia, Cheong-Hee Changb, Donna L. Farbera,*
aDivision of Transplantation, Department of Surgery, University of Maryland School of Medicine, MSTF Building, Room 400,
Baltimore, 685 W. Baltimore St., Baltimore, MD 21201, USAbDepartment of Microbiology and Immunology, Indiana University, Indianapolis, IN, USA
Received 15 May 2003; received in revised form 29 July 2003; accepted 29 July 2003
Abstract
It has been difficult to assess the role of specific genes in activation and differentiation of peripheral T cell subsets such as
naive, effector and memory T cells due to the impairments in T cell development and immune pathologies often observed in
genetically manipulated mouse models, and the lack of reliable methods for introducing genes into primary mouse T cells. In
this study, we demonstrate transient transfection of genes into resting and activated mouse CD4 T cell subsets using
‘‘Nucleofectionk’’, a modified electroporation technique. Using this approach, cDNA encoding green fluorescent protein
(GFP) is efficiently taken up and expressed by purified polyclonal and antigen-specific mouse naive, effector and memory CD4
T cells isolated from BALB/c or TCR-transgenic mice. The resultant transfected resting T cells are fully amenable to TCR-
mediated activation. We also demonstrate that expression of endogenous gene can be turned on in resting T cells by transfection
of a transcriptional transactivator. Our results demonstrate for the first time, the expression of exogenously transfected genes
and the modulation of endogenous gene expression in primary mouse T cell subsets. This technology will enable a variety of
mechanistic questions on T cell activation, function and signaling to be addressed in T cells that differ in activation history and
functional capacities.
D 2003 Elsevier B.V. All rights reserved.
Keywords: T lymphocytes; Rodent; Cellular differentiation; Molecular biology; Gene therapy
1. Introduction
Manipulation of gene expression in T cell lines and
clones by the introduction, deletion, or mutation of
specific genes, has enabled mechanistic dissection of
0022-1759/$ - see front matter D 2003 Elsevier B.V. All rights reserved.
doi:10.1016/j.jim.2003.07.015
Abbreviations: GFP, green fluorescent protein; HA-TCR,
hemagglutinin-specific T cell receptor transgenic mouse.
* Corresponding author. Tel.: +1-410-706-7458; fax: +1-410-
706-0311.
E-mail address: [email protected] (D.L. Farber).
molecular requirements for T cell activation, signaling
and function. However, these cultured T cells do not
represent the distinct differentiation states that T
lymphocytes exist in vivo, namely naive, effector
and memory subsets. In vivo modulation of gene
expression using transgenic and targeted knock-out/
knock-in technology has established the roles of
specific genes in T cell development and function,
although the majority of these genetic modulations
either abrogate T cell development (Molina et al.,
1992; Negishi et al., 1995; Clements et al., 1998),
W. Lai et al. / Journal of Immunological Methods 282 (2003) 93–10294
impair peripheral naive T cell activation and differen-
tiation (Peng et al., 2001), and/or result in large-scale
immune pathologies (Chambers et al., 1997; Majeti et
al., 2000). Therefore, the molecular events controlling
peripheral T cell differentiation into effector and
memory T cell subsets, and comparing the role of
specific genes in naive, effector and memory T cell
function have not been possible to assess using mouse
genetic models.
In order to circumvent the limitations of global in
vivo gene manipulation, yet address mechanistic
questions on peripheral T cell differentiation and
function, it would be advantageous to introduce genes
into mouse primary T cell subsets in distinct differen-
tiation and/or activation states. However, approaches
for transfecting mouse T cells have been adapted only
for activated T cells, and transfection of resting naive
or memory T cell subsets has not been demonstrated.
Murine leukemia virus-based vectors have been used
to transfect genes into in primary mouse T cells
activated with mitogens, anti-CD3/anti-CD28 anti-
bodies, or antigen (Randolph et al., 1999; Van Parijs
et al., 1999; Walker and Green, 1999; Burr et al.,
2001), resulting in infection efficiencies ranging from
10% to 20% (Burr et al., 2001), and 40% to 90% (Van
Parijs et al., 1999). There are significant limitations to
this approach, however, as resting T cells are not
permissive to infection by murine leukemia viruses,
and the efficiency of gene uptake using retroviral
vectors critically depends on the extent of T cell
activation (Hagani et al., 1999; Van Parijs et al.,
1999). Lentiviral-based vectors can transduce DNA
into resting primary human T lymphocytes (Chinnas-
amy et al., 2000), and into mouse embryos for the
generation of transgenic mice (Lois et al., 2002),
although their use in primary mouse T cells ex vivo
has not yet been demonstrated.
Non-retroviral-mediated gene delivery systems,
such as electroporation, avoid the biohazards associ-
ated with using recombinant retroviruses, yet these
methods have been more widely adapted to transfec-
tion of primary human T cells. Electroporation has
been shown to efficiently introduce DNA into acti-
vated and freshly isolated human T cells (Hughes and
Pober, 1996; Solomou et al., 2001a,b), with transfec-
tion efficiencies of 32% in primary human T cells
(Herndon et al., 2002). By contrast, resting mouse T
cells are resistant to DNA uptake via conventional
electroporation (Cron et al., 1997; Bell et al., 2001),
although preactivation of mouse T cells with anti-TCR
antibodies resulted in efficient gene transfection (Bell
et al., 2001).
In this study, we demonstrate the efficient delivery
of genes into purified resting and activated mouse
CD4 T cell subsets using a modified electroporation
technique, termed Nucleofectionk (Amaxa Biosys-
tems, Cologne, Germany). Nucleofection of resting
CD4 T cells with cDNA encoding the green fluores-
cent protein resulted in transfection efficiencies up-
wards of 20–25%, and the resultant transfected
resting T cells were fully amenable to TCR-mediated
activation. We demonstrate that sorted naive
(CD45RBhi) and antigen-specific naı̈ve CD4 T cells
isolated from BALB/c and TCR-transgenic mice,
respectively, exhibit transfection efficiencies of 6–
12%, whereas resting memory (CD45RBlo) CD4 T
cells exhibit substantially higher transfection efficien-
cies than naı̈ve counterparts (23–25%), and effector
cells exhibit the highest transfection efficiency (35%).
Finally, we demonstrate that an endogenous gene can
be selectively turned on in resting T cells by trans-
fection of the appropriate transcriptional activator. Our
results demonstrate for the first time, the expression of
exogenously introduced genes into primary resting
subsets of mouse T cells, and set the stage for
mechanistic studies investigating the role of specific
genes in T cell subsets that differ in activation and
differentiation state.
2. Materials and methods
2.1. Mice
BALB/c mice between 6 and 8 weeks of age were
obtained from the National Cancer Institute Biological
Testing Branch. HA-TCR transgenic mice expressing
the TCR specific for influenza hemagglutinin peptide
and I-Ed (Kirberg et al., 1994) were bred and main-
tained at the Animal Care Facility at the University of
Maryland Medical School, Baltimore, MD.
2.2. Antibodies, plasmids and reagents
The following Abs were purified from culture
supernatants from hybridomas maintained in the lab-
W. Lai et al. / Journal of Immunological Methods 282 (2003) 93–102 95
oratory as described previously (Ahmadzadeh et al.,
1999): anti-CD3q (C363. 29B), anti-CD4 (GK1.5),
anti-CD8 (TIB 105) and Anti-I-Ad (212.A1). PE-
conjugated anti-CD45RB (clone 363.16A), allophy-
cocyanin (APC)-coupled anti-CD4 and PE-conjugated
anti-CD25 were obtained from BD-Pharmingen (San
Diego, CA). Nucleofectork Kit V, provided by
Amaxa Biosystem (Amaxa, Cologne, Germany),
was used for primary cell transfection. In all cases,
cells were cultured in Complete Click’s Medium
(Santa Ana, CA) containing 5% Fetal Bovine Serum,
1% L-glutamine-200 mM (GIBCO/BRL), 1% Penicil-
lin–Streptromycin (GiBCO BRL), 1% 1 M Hepes
buffer (Gibco/BRL) and 0.056 mM 2-mercaptoetha-
nol (Sigma, St. Louis, MO). HA peptides 110–119 of
the sequence, SFERFEIFPK, were synthesized by the
Biopolymer Laboratory, University of Maryland
School of Medicine. The pCMV-EGFPN1 vector
(Clontech) used for transfection was provided by Dr.
Ferenc Livak, Department of Microbiology and Im-
munology, University of Maryland, Baltimore. The
plasmid construct pCIITA (Sisk et al., 2001) contain-
ing the MHC Class II transactivator (CIITA) under
control of the CMV promoter was generated by
cloning the full-length CIITA cDNA into the multiple
cloning site of pcDNA3 (Invitrogen, Carlsbad, CA).
2.3. Isolation of CD4 T cell subsets
The isolation of enriched populations of splenic
CD4 T cells (85–90% pure) from BALB/c and HA-
TCR mice, has been described previously (Farber et
al., 1995; Ahmadzadeh et al., 1999). For generation of
effector CD4 cells, Balb/c or HA-TCR CD4 T cells
(1�106 cells/ml) were incubated with 5 Ag/ml anti-
CD3 antibody (C29B.363) or HA peptide, respective-
ly, in the presence of mitomycin C-treated (Roche,
Indianapolis, IN) T-depleted splenic APC isolated as
previously described (Ahmadzadeh et al., 2001)
(3� 106 cells/ml) for 2–3 days at 37 jC. The resul-
tant activated/effector cells were centrifuged through
Ficoll, washed in PBS to remove dead and contami-
nating accessory cells and re-suspended in complete
Click’s medium, resulting in 95% pure effector CD4 T
cells. CD4 T cells were also fractionated into purified
naive (CD45RBhi) and memory (CD45RBlo) CD4 T
cells by automated magnetic separation using the
AutoMACSk system (Miltenyi Biotec, Auburn,
CA) as previously described (Hussain et al., 2002).
Briefly, CD4 T cells were incubated with PE-conju-
gated Anti-CD45RB Ab (Pharmingen) followed by
anti-PE magnetic microbeads (Miltenyi Biotec). The
cells were separated by automated passage over a
ferromagnetic column followed by serial elutions
resulting in CD45RBlo (>99% pure) and CD45RBhi
(>98% pure) populations.
2.4. Transfection of murine T cells using Nucle-
ofectionk
Cells prepared as described above were washed
once in PBS and 3� 106–4� 106 cells were resus-
pended in 100 Al Nucleofectork Solution V (‘‘Kit
V’’, Amaxa Biosystems) warmed to room tempera-
ture, and 5–10 Ag plasmid DNA (pCMV-EGFPN1)
was immediately added and mixed well. The cell–
DNA mixture was subsequently transferred to an
electroporation cuvette with aluminum electrodes
(Amaxa) and placed in the Nucleofectork device
(Amaxa). Nucleofection of the cells was accom-
plished using the T-27 program, and samples were
immediately transferred to 24-well plates containing
1 ml pre-warmed media. Cells were incubated at
37jC, 5% CO2 for 16–48 h and analyzed by flow
cytometry and microscopy.
2.5. FACS analysis and fluorescent microscopy
Cells were washed once in Stain buffer (PBS/
3%FCS/0.1% sodium azide), and stained with PE-
conjugated anti-CD4, anti-CD45RB, or allophycocya-
nin-conjugated anti-CD4, PE-conjugated anti-I-A/I-E
(all from BD-Pharmingen). The 6.5 anti-TCR clono-
type antibody (Kirberg et al., 1994) was conjugated to
biotin and Allophycocyanin-conjugated streptavidin
(BD-Pharmingen) was used for FACS staining.
Stained cells were analyzed using the FACScaliburk(Becton Dickinson, San Jose, CA) and CellQuestksoftware. For visualization of transfected cells by
fluorescence microscopy, 106 cells were suspended
in 100 Al of complete Click’s medium and plated on
poly-L-lysine coated slides for 1 h on ice. Cells were
washed twice with PBS, air-dried and samples were
analyzed by fluorescence microscopy using the Zeiss
Axioskop (Zeiss, Germany) with Axiovision 3.0
software.
W. Lai et al. / Journal of Immunological Methods 282 (2003) 93–10296
3. Results
3.1. Optimization of nucleofection conditions for
resting primary mouse CD4 T cells
Purified mouse resting CD4 T cells are resistant
to gene transfection by conventional retroviral vec-
tors (Van Parijs et al., 1999) and electroporation
(Cron et al., 1997). Because nucleofection technol-
ogy (Amaxa) targets DNA directly to the cell
nucleus independent of cell cycle, we asked whether
nucleofection could be adapted to mediate efficient
DNA uptake by transfection-resistant primary mouse
CD4 T cells. We isolated splenic CD4 T cells ex
vivo from unmanipulated BALB/c mice as described
previously (Ahmadzadeh et al., 1999), and assayed
their ability to take up and express cDNA encoding
the green fluorescent protein (GFP), using a number
of different nucleofector solutions and programs
available from the manufacturer, whose efficacy
with primary mouse T cells had not been tested.
As shown in Fig. 1A, while a low proportion of
mouse CD4 T cells are transfected when pulsed
with the U-14 program optimized for human T cells,
a substantial proportion (21%) of CD4 T cells
express the transfected DNA using the T-27 pro-
gram. The non-CD4 T cells remaining after the CD4
cell enrichment procedure (comprising mostly resid-
ual granulocytes, macrophages and a small propor-
tion of B cells, data not shown) are not amenable to
gene transfection using these nucleofection condi-
tions (Fig. 1). These results indicate that resting
mouse CD4 T cells are amenable to DNA uptake
when administered with the appropriate solution and
electric field conditions. Cell viability using these
nucleofection conditions was consistently >75%
(data not shown), and transfection efficiencies of
>20% with purified CD4 T cells were consistently
achieved.
We next assessed the stability of gene expression
by resting CD4 T cells at time points of 16–36-
h post-nucleofection. We found that expression
peaked at 16-h post-transfection and diminished
over time to 38% of the peak expression levels
by 36 h (Fig. 1B). We also detected equivalent
gene expression from 8 to 16 h (data not shown).
To ensure that the cells were expressing sufficient
amounts of GFP for visualization, we analyzed the
untransfected and transfected populations by fluo-
rescent microscopy. The CD4 T cells that take up
and express GFP are clearly visible by green
fluorescence (Fig. 1C). These results indicate that
gene transfection by nucleofection can enable mi-
croscopic analysis of the effect of a transfected
gene.
3.2. Transfected resting CD4 T cells are amenable to
TCR/CD3-mediated activation
Although the viability of the transfected cells
was high (>75%), it was possible that the transfec-
tion procedure itself, and/or expression of an in-
nocuous gene by transfection would compromise
the functional capacity of the T cells. To address
this concern, we assessed the ability of transfected
CD4 T cells to become activated via anti-CD3
cross-linking. For these experiments, resting CD4
T cells transfected with pEGFP as in Fig. 1 were
either cultured in media alone, or on plates con-
taining immobilized anti-CD3 antibody for 24 or 48
h, for TCR/CD3-mediated cross-linking and activa-
tion. As shown in Fig. 2, GFP was expressed in
nucleoporated cells cultured in media alone for 24
h or in the presence of anti-CD3 cross-linking for
24 or 48 h (top row), although the brightness of
green fluorescence was decreased at 48 h. We
assessed the ability of untransfected versus trans-
fected cells to be activated by assessing upregula-
tion of IL-2 receptor (CD25) expression in GFP�
versus GFP+ cells, respectively (Fig. 2). While
resting T cells typically exhibit 12% CD25 expres-
sion, 24- and 48-h activated cells showed CD25
upregulation on 25% and 70–80% of CD4 T cells,
with equivalent levels of activation-induced CD25
upregulation observed in untransfected (GFP�) and
transfected (GFP+) CD4 T cells (Fig. 2, second and
third row). These results demonstrate that gene
transfection of resting CD4 T cells does not com-
promise their ability to be activated via the TCR/
CD3 complex.
3.3. Gene transfection of resting and activated CD4 T
cell subsets
Because peripheral T cells exist in heterogeneous
differentiation states, such as naı̈ve, effector and
Fig. 1. Transfection of resting CD4 T cells. CD4 T cells were purified from BALB/c splenocytes, resuspended in Nucleofectork solution V, and
placed in a cuvette with aluminum electrodesF 10 Ag pCMV-EGFP-N1 plasmid (Clontech) encoding the green fluorescent protein (GFP), and
subject to electroporation using the Nucleofectork device. (A) Expression of GFP by electroporated CD4 T cells using two different
Nucleofectork programs. The percentage of CD4+GFP+ cells is indicated in the upper right quadrant. (B) Kinetics of expression. GFP
expression 16–36 h following nucleofection. Controls with no DNA after 24 and 36 h showed no GFP expression (data not shown). (C)
Fluorescent microscopy of GFP expression in transfected versus control CD4 T cells. The results in this figure are representative of six separate
experiments.
W. Lai et al. / Journal of Immunological Methods 282 (2003) 93–102 97
memory T cells, we wished to assess whether
nucleoporation would enable gene transfection into
these multiple subsets. First, we asked whether the
ability of resting CD4 T to be transfected was due
to DNA uptake by both naive and memory subsets,
and whether one subset was more efficiently trans-
fected. We therefore fractionated resting splenic
CD4 T cells isolated as in Fig. 1 on the basis of
CD45RB isoform expression, with CD45RBhi de-
lineating naive cells and CD45RBlo representing
memory cells (Bottomly et al., 1989; Lee and
Vitetta, 1991). As shown in Fig. 3, both purified
naive (CD45RBhi) and memory (CD45RBlo) CD4
T cell subsets can be transfected via nucleofection,
although the transfection efficiency is lower for
sorted naive T cells (6–10%) (left FACS plots),
Fig. 2. GFP-Transfected resting T cells are amenable to TCR/CD3-mediated activation. CD4 T cells isolated as above were transfected with
pCMV-EGFP-N1 and either cultured in media alone, or cultured in the presence of immobilized anti-CD3 antibody for 24 and 48 h. GFP
expression of unstimulated and stimulated cells is shown on the top row, with the percent CD4+GFP+ indicated in the upper right quadrant.
CD25 expression gated on untransfected (CD4+GFP�; second row) and transfected (CD4+GFP+; third row) cells. These results are
representative of three experiments.
W. Lai et al. / Journal of Immunological Methods 282 (2003) 93–10298
as compared to memory CD4 T cells (23% GFP+,
right FACS plots). These results demonstrate that
while both resting naive and memory subsets are
amenable to gene uptake, memory T cells can be
transfected with greater efficiency. Furthermore, our
ability to transfect unfractionated CD4 T cells with
higher efficiency than purified naive cells reflects the
contribution of the higher transfection efficiency of
memory T cells, which comprise 20% of total CD4
T cells in conventionally housed 8–12-week-old
BALB/c mice (data not shown).
Second, we wanted to assess whether the trans-
fection efficiency changed with activation by poly-
clonal TCR/CD3-mediated stimulation or by antigen.
As shown in Fig. 4A, CD4 T cells polyclonally
activated with anti-CD3 and antigen-presenting cells
(APC) exhibited increased transfection efficiency
compared to resting cells, and the efficiency was
further augmented by prolonged activation time.
While resting CD4 T cells were transfected with an
efficiency of 14.7% in a representative experiment,
this efficiency increased to 24% and 35% when the
cells were pre-activated for 2 and 3 days, respec-
tively (Fig. 4A). These results indicate that activated
CD4 T cells readily take up and express transfected
DNA with an efficiency that exceeds that of resting
CD4 T cells.
We also used an antigen-specific TCR transgenic
system to more precisely establish the transfection
efficiency of naive versus activated, antigen-specific
CD4 T cells. We isolated CD4 T cells from HA-TCR
T cell receptor transgenic mice, in which 30–40% of
Fig. 3. Transfection of naive and memory CD4 T cells. Naive (CD45RBhi) and memory (CD45RBlo) CD4 T cells were fractionated using the
AutoMACS (see Materials and methods), and subject to nucleofection as above. Percentages of CD4+GFP+ cells are indicated and these data are
representative of three experiments.
Fig. 4. Transfection of resting and activated CD4 T cells. (A) Transfection of polyclonally activated T cells. Purified CD4 T cells were either
transfected with pCMV-EGFP or activated for 2–3 days with anti-CD3 antibody in the presence of splenic antigen-presenting cells (see
Materials and methods) prior to transfection. The percentage of CD4+GFP+ T cells is indicated in the upper right quadrant. (B) Transfection of
resting and activated antigen-specific T cells. CD4 T cells were purified from HA-TCR transgenic mice and nucleofectedF pEGFP as above,
cultured overnight and stained with the 6.5 anti-TCR clonotype antibody (upper row). HA-TCR CD4 T cells were activated in vitro with HA
peptide (1 Ag/ml) and mitomycin-C treated BALB/c APC for 3 days, nucleofected with pEGFP and analyzed following an overnight culture
(‘‘HA-Act’’, lower). These results are representative of three experiments.
W. Lai et al. / Journal of Immunological Methods 282 (2003) 93–102 99
Fig. 5. Upregulation of MHC Class II expression in resting CD4 T
cells following transfection of the CIITA transcriptional activator.
CD4 T cells were isolated as above and nucleofected as above either
with pCIITA containing the full-length cDNA for the MHC Class II
transactivator driven by the CMV promoter or with a non-CIITA
control plasmid (pEGFP). FACs plots show CD4 versus MHC Class
II expression on control and pCIITA-transfected cells. Numbers
indicate absolute percentages of cells in each quadrant.
W. Lai et al. / Journal of Immunological Methods 282 (2003) 93–102100
CD4 T cells express a clonotypic TCR (clonotype 6.5)
specific for influenza hemagglutinin (HA) and I-Ed
(Kirberg et al., 1994) and a naive phenotype (Ahmad-
zadeh et al., 1999). When transfected with the GFP-
encoding plasmid, nearly 13% of freshly isolated HA-
TCR CD4 T cells are GFP+ (Fig. 4B), which also
represents 37% of the 6.5+ CD4 T cells. We previ-
ously reported that activation of HA-TCR CD4 T cells
with the cognate antigenic HA peptide and APC
results in differentiation to effector CD4 T cells that
are uniformly 6.5+, and exhibit all of the effector-
specific functional and phenotypic attributes (Ahmad-
zadeh et al., 1999, 2001). Fig. 4B shows that these
antigen-activated effector CD4 T cells can likewise be
efficiently transfected. These results demonstrate that
antigen-specific naive and effector T cells are amena-
ble to gene uptake, enabling gene manipulations in an
antigen-specific system.
3.4. Modulation of endogenous gene expression
Having demonstrated the consistent expression of
an exogenously introduced gene in primary mouse T
cells, we wished to assess the functional capacity of a
transfected gene in resting T cells and also whether
nucleofection could be used to modulate endogenous
gene expression by the introduction of transcriptional
modulators. To explore these issues, we transfected
cDNA encoding a transcriptional activator, the MHC
Class II transactivator (CIITA), into resting T cells and
assayed for expression of the endogenous target gene,
MHC Class II. CIITA has been shown necessary and
sufficient for MHC Class II expression in cell lines
(Chang et al., 1995) and in transgenic mouse models
(Otten et al., 2003), and was therefore a good model
gene to test in our assay. Transfection of a plasmid
encoding CIITA under the control of the CMV pro-
moter into freshly isolated mouse splenic CD4 T cells
resulted in upregulation of MHC Class II expression
in 13–15% of CD4 T cells (Fig. 5, right), compared to
a lack of MHC Class II expression in either mock-
transfected (data not shown), or CD4 T cells trans-
fected with control pEGFP plasmid (left FACS plot).
Contrary to the lack of transfection of non-CD4 T
cells with pEGFP, a proportion of non-CD4 T cells
appeared to be transfected with pCIITA and express
MHC Class II (1% total cells and 17% of non-CD4 T
cells) (Fig. 5, right). These results demonstrate func-
tional competence of a transfected gene product, and
that endogenous gene expression in resting T cells can
be modulated by transcriptional activators.
4. Discussion
Primary mouse T cells are notoriously resistant to
gene uptake by retroviral and non-retroviral methods,
and transfection of T cell subsets differing in func-
tional capacities and activation history has not previ-
ously been accomplished. Here, we demonstrate
efficient gene uptake of resting and activated murine
CD4 T cell subsets using a modified electroporation
technique termed nucleofection. Using this approach,
we were able to achieve transfection efficiencies of
20% with resting CD4 T cells and 35% with activat-
ed/effector CD4 T cells. Moreover, the transfection
procedure and the expression of an exogenous gene
did not compromise their ability to be activated via
the cell surface TCR. When fractionated into resting
naive and memory subsets, we found that memory
CD4 T cells were more permissible to transfection,
with efficiencies of 23%, whereas naive T cells
exhibited 6–12% transfection efficiency. We also
demonstrate that transfection of the transcriptional
activator CIITA can turn on expression of its endog-
enous target gene, MHC Class II in resting CD4 T
cells. Our results provide the first evidence for
efficient uptake, expression and functional compe-
tence of transfected genes in primary subsets of
mouse CD4 T cells.
W. Lai et al. / Journal of Immunological Methods 282 (2003) 93–102 101
The ability to introduce genes into resting and
activated mouse T cell subsets now enables mecha-
nistic studies on function, signaling, and transcrip-
tional regulation in naive, effector, and memory T
cells, that cannot be accomplished using in vivo
mouse transgenic and knockout models. For example,
mice deficient in key signaling intermediates for T cell
activation such as tyrosine kinases (Molina et al.,
1992; Negishi et al., 1995), linker/adapter molecules
(Clements et al., 1998; Zhang et al., 1999), MAP
kinases (Pages et al., 1999) and critical transcription
factors (Oukka et al., 1998; Peng et al., 2001) either
lack peripheral T cells or exhibit profound peripheral
T cell dysfunctions, and are therefore not useful for
assessing the roles of these molecules in wildtype
peripheral T cell differentiation. With this nucleofec-
tion approach, the role of specific genes in wildtype
naive, effector and memory T cell function can be
directly assessed, without potential complications of
in vivo dysfunctions found in genetically altered
mouse T cells.
Our results demonstrate that different subsets of
resting T cells are not equivalently permissible to gene
transfection. Although the memory (CD45RBlo) sub-
set is comprised of small, resting cells that do not
express CD25 (Farber et al., 1995), we consistently
achieved higher transfection efficiencies with resting
memory as compared to resting naive T cells
(CD45RBhi or antigen-specific naı̈ve T cells from
HA-TCR mice). As it has been demonstrated that a
fraction of these memory phenotype CD4 T cells
undergo cycling in vivo (Tough and Sprent, 1994),
the increased transfection efficiency may be due to the
low level cycling of some memory T cells. Although
we found the transfection efficiency of naive CD4 T
cells averaged 10%, this proportion still provides
sufficient numbers for subsequent sorting of positive
cells, particularly as cell viability routinely exceeds
75%.
While our initial analysis focused on transfection
of an exogenous gene not normally expressed in
mammalian cells, we also wanted to assess whether
transfected gene products could function appropriate-
ly in primary resting T cells. We chose to examine
transfection of the transcriptional activator, CIITA for
two reasons: first, to assess the functional competence
of a transfected gene product in primary T cells, and
second, to determine if transient transfection could be
used to modulate expression of endogenous genes in
resting T cells. Our findings that CIITA can activate
MHC Class II expression in resting CD4 T cells
demonstrate the utility of this approach for modulat-
ing gene expression in resting T cells by transcrip-
tional activation. CIITA is not expressed in resting
human or mouse T cells (Wong et al., 2002; Otten et
al., 2003), but has been shown to be upregulated in
activated human T cells (Wong et al., 2002) and by IL-
12 induction in activated mouse Th1 cells (Gourley et
al., 1999), but not in anti-CD3 activated mouse T cells
(Otten et al., 2003). Our results establish the suffi-
ciency of CIITA for driving MHC Class II expression
in mouse T cells in the absence of T cell activation.
In summary, we demonstrate for the first time,
efficient gene delivery and expression in resting and
activated subsets of mouse CD4 T cells, enabling the
design of mechanistic experiments to identify how
distinct functions and pathways are regulated in T cell
subsets of diverse activation and differentiation states.
Acknowledgements
We wish to thank Dr. Gregg Hadley (Department
of Surgery, UMB) for critical reading of this manu-
script. This work was supported by NIH AI42092
awarded to D.L.F.
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