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,
*
Department of Medicine, Division of Immunology,
Department of Molecular Pharmacology, and
Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford CA 94305
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Varied approaches to alter or abrogate inflammatory T cell responses in autoimmune disease states have been explored. Among the most promising are those that exploit T cells for delivery of therapeutic proteins (18, 19, 20, 21). Early studies from our laboratory, using an animal model of the human disease multiple sclerosis, experimental autoimmune encephalomyelitis (EAE), demonstrated that autoantigen-specific T cell hybridomas, transduced using retroviral vectors containing genes encoded for regulatory cytokines were capable of delivering cytokines to the autoimmune lesions in the CNS (22, 23). Disease could be ameliorated or exacerbated depending on the cytokine that was delivered. Transduced T cell hybridomas, which lacked the tissue-specific TCR for Ag but secreted the same anti-inflammatory cytokines, were ineffective. Thus, tissue-specific homing and/or retention was required for therapeutic effect. More recent studies have used transduced autoantigen-specific T cell clones to deliver immunosuppressive proteins to autoimmune lesions of EAE (24). These studies demonstrated that expression of retroviral-encoded regulatory cytokines did not alter the homing abilities of transduced T cell hybridomas or T cell lines, and, importantly, demonstrated that T cells, transduced by retrovirus for the expression of regulatory genes, had the potential to deliver these proteins in a site-specific manner. If nontransformed, primary T cells could be used to replace T cell hybridomas and T cell lines, then transduction of autoantigen-specific T cells would represent a viable therapeutic strategy for the treatment of autoimmune disease.
Primary murine CD4+ T cells have been extremely difficult to infect with retroviruses, and are more often refractory to retroviral transduction. However, data presented in this paper demonstrate efficient and robust retroviral transduction of Ag-specific murine CD4+ T cells from naive TCR transgenic or conventional Ag-challenged mice using the pGCIRES vector. Studies using "conventional" mice primed with foreign peptide confirmed that the small population of Ag-specific CD4+ T cells could be targeted by retroviruses for transduction and then isolated using the pGCIRES marker protein, GFP (green fluorescent protein).3 The development of stable retroviral-mediated transduction of autoantigen-specific murine CD4+ T cells should provide a novel method to alter T cell function and is of particular interest for the application of gene therapy in autoimmune disease.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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PL/J and DBA/2 mice were purchased from The Jackson Laboratory (Bar Harbor, ME). B10.PL mice, transgenic for a TCR reactive with myelin basic protein (MBP) NAc1–11, have been described elsewhere (25) and were obtained from Dr. Caroline C. Whitacre (Ohio State University, Columbus, OH). Mice were used between 6 and 12 wk of age and maintained in the Department of Comparative Medicine, Stanford Medical Center.
Plasmids
The pGCIRES retroviral plasmid was constructed using the
pLCAT backbone with the 5' and 3' long terminal repeats (LTR)
and the psi (
) packaging sequence from the
Moloney murine leukemia virus (MMLV)-based MFG vector (provided by Dr.
R. C. Mulligan). A 722-bp enhanced codon-substituted humanized
form of green fluorescent protein (huGFP) with site-specific mutations
(S66T, V164A, H232L, and a G to A base change at residue 69) to the
native jellyfish (Aequoria victoria) protein was used as the
reporter protein (provided by Dr. M. A. Anderson, Stanford
University) and was inserted into the retroviral plasmid by
BamHI and NcoI digestion (New England Biolabs,
Beverly, MA). The encephalomyocarditis virus internal ribosome entry
site (EMCV IRES) from pWZLBleo (provided by P. J. Morgenstern) was
cloned upstream of the GFP by SnaBI/NcoI to
PmlI/NcoI (insert to vector) restriction
ligation. The multiple cloning site from the pCR-Script Amp plasmid
(Stratagene, La Jolla CA) was amplification with standard T3 and T7
oligonucleotide primers and cloned upstream of the EMCV IRES into the
T4 DNA polymerase-treated SalI site. The final
ampicillin-resistant bicistronic retroviral reporter construct was 6691
bp and termed pGCIRES. The pWTGFP plasmid is a murine stem cell
virus-based retroviral vector and contained the puromycin resistance
gene, polio IRES, and wild-type GFP. The plasmid Ca116 mp60hg4
contained the murine p60 TNF receptor (TNFR) subunit linked to the
human IgG4 Fc domain provided by Dr. Rachael Ettinger (Stanford
University, Stanford CA) and Dr. J. Browning (Biogen, Cambridge
MA). The soluble TNFR (sTNFR) fragment (1381 bp) was subcloned into
pcDNA3 (Invitrogen, Carlsbad, CA) and was obtained by
EcoRV/XhoI digestion and directionally cloned
into pGCIRES between the SrfI and XhoI sites
and termed pGCIRES.sTNFR (8072 bp). The murine IL-4 gene (447 bp)
was amplified by PCR and subcloned into pGCIRES using SrfI
restriction ligation and termed pGCIRES.IL4 (7138 bp).
Cell lines
The Phoenix retroviral producer cell lines are derived from a 293T cell line and have been described elsewhere (26). Ecotropic packaging cells (Phoenix-E) were cultured in DMEM complete (DMEM-C) that contained DMEM (Life Technologies, Gaithersburg MD) supplemented with 10% FCS, 2 mM glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin. Retroviral producer lines were cultured in DMEM-C medium that contained diphtheria toxin (1 µg/ml) (Calbiochem, La Jolla, CA) for selection of the ecotropic envelope gene and hygromycin B (300 µg/ml) (Boehringer Mannheim, IN) for selection of the gag-pol genes. The NIH 3T3 cell line (ATCC CCL92; American Type Culture Collection, Manassas, VA) was cultured in DMEM supplemented with 10% bovine serum (JRH Biosciences, Lenexa KS), 2 mM L-glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin. All cells were maintained at 37°C in 6% CO2.
Transfection assay
A total of 2.0 x 106 Phoenix-E packaging cells were cultured in 12 ml DMEM-C in 100-mm tissue culture dishes (Nalge Nunc International, Roskilde, Denmark). Following overnight incubation, the packaging cells were transfected with 10 µg retroviral plasmid DNA (Qiagen, Valencia, CA) or no plasmid DNA (mock) using a modified version of the calcium phosphate precipitation protocol described elsewhere (26). At 8–12 h posttransfection, calcium phosphate-containing medium was replaced with DMEM-C medium and cultures were maintained at 37°C in 6% CO2 for 24–48 h, then at 32°C in 6% CO2 for 16–24 h. Viral supernatant from transient transfections was harvested and filtered using a 0.45 µm filter (Nalge, Rochester NY), then stored at -80°C. Virus titers were determined for the Phoenix-E retroviral packaging cell lines using NIH 3T3 lines, as previously described (27). Virus stocks with titers >4 x 106 were used for transduction of murine cells.
Ag-specific CD4+ populations
Peptides of sperm whale myoglobin (SWM, 110–121) (AIIHVLHSRHPG) and MBP (NAc1–11) (NAc-ASQKRPSQRHG) were synthesized and HPLC purified at the Protein and Nucleic Acid Facility (Beckman Center, Stanford University). Immunization of DBA/2 mice for production of Ag-reactive CD4high cells was conducted as previously described (28). Briefly, groups of 5–10 mice were immunized s.c. at the base of the tail with 100 µg SWM peptide in 100 µl IFA plus 10 µg/ml heat-killed Mycobacterium tuberculosis H37RA (Difco Laboratories, Detroit MI). At 8–10 days (or as indicated) postimmunization, single cell suspensions (2.0–5.0 x 106 cells/ml) from either spleen or draining inguinal lymph nodes were cultured for transduction or stimulated in vitro before transduction for time periods indicated with Ag (10 µM SWM). Single cell splenocyte preparations (5.0 x 106 cells/ml) from MBP TCR transgenic mice were stimulated in vitro for 24 h with Ag (MBP NAc1–11, 6 µM) or PMA (50 ng/ml) and ionomycin (1 µM). T cell medium (RPMI complete, RPMI-C) consisted of RPMI 1640 (Life Technologies) supplemented with 2 mM L-glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin, 1% nonessential amino acids, 1 mM sodium pyruvate, 10 mM HEPES buffer, 50 µM 2-ME, and 10% FCS (HyClone, Logan UT). Cells were maintained at 37°C with 6% CO2.
Infection assay
A total of 2.0–5.0 x 106 cells in 0.5 ml RPMI-C were plated in duplicate in 6-well plates (Falcon Labware, Franklin Lakes, NJ) and overlaid with 2 ml thawed recombinant retroviral supernatant supplemented with protamine sulfate (8 µg/ml) (Sigma, St. Louis MO). Plates were centrifuged at 2500 rpm at 32°C for 2 h and transferred to incubation at 32°C in 6% CO2 for 16 h, or at time periods indicated. Medium was exchanged with RPMI-C medium supplemented with 10 U/ml murine recombinant IL-2 (R&D Systems, Minneapolis, MN) and then transferred to 37°C in 6% CO2 for an additional 24 h. At 48 h postinfection, cells were analyzed for transduction by flow cytometric analysis of GFP expression.
FACS
Murine cells transduced with recombinant retrovirus cells were stained with rat anti-mouse CD4-PE (Caltag Laboratories, San Francisco CA). Dead cells were excluded using propidium iodide (PI) staining (Sigma) and live cells were analyzed for CD4-PE (PE channel) and GFP reporter expression (FITC channel). Analysis was performed on a FACScan cytometer (Becton Dickinson, Mountain View, CA). For CD4high T cell analyses, cells were stained with CD4-PE and sterile sorted into CD4normal or CD4high populations using a FACStar flow cytometer (Stanford FACS Facility, Stanford University). Sorted sample populations were reanalyzed following the initial collection to confirm sample fraction purity and CD4 expression (>95%). All data were analyzed using FlowJo (version 2.7.8) flow cytometry software (Tree Star, San Carlos CA).
Cell cycle analysis
For nonvital cell cycle analyses, cell samples (1–3 x 106) were pelleted and resuspended in 200 µl buffer containing PBS and 2% FCS (FACS buffer). Single cell suspensions were resuspended in chilled 70% ethanol (2 ml) and incubated at least 18 h at 4°C for fixation. Cells were then pelleted and resuspended by passing through a 25-gauge needle into 800 µl of FACS buffer containing 100 µg/ml RNase and 40 µg/ml PI. Cell cycle distributions were analyzed by flow cytometry using linear scales with PI staining as a measure of DNA content. For vital cell cycle analyses, the cell samples were pelleted and resuspended in FACS buffer (1 ml) and stained with rat anti-mouse CD4-PE at a 1:100 dilution (Caltag Laboratories) for 30 min on ice. Hoechst 33342 (10 µg/ml) (Molecular Probes, Eugene OR) and verapamil (50 µg/ml) (Sigma) was added before incubation at 37°C for 1 h. Following incubation, samples were transferred to ice and kept in the dark before analysis using flow cytometry. Cells were analyzed and sorted using linear scales, and DNA content was measured by monochlorobimane and Hoechst 33342 lasers. The cells were fractionated into three populations: G0/G1, S+G2/M, or M. The median third (33%) of the G0/G1 and S+G2/M populations and distal third (66–99%) of the M population was collected (5 x 105 cells collected per fraction). Sorted sample populations were reanalyzed following the initial collection to confirm sample fraction purity and CD4 expression (>95%).
ELISA analysis
IL-4 production was measured by a standard sandwich ELISA protocol. Supernatants were harvested 48 h after in vitro stimulation with 5 µg/ml MBP NAc1–11. Capture anti-IL-4 Ab (11B11, 1 µg/ml) (PharMingen, San Diego, CA) was incubated in 96-well Maxisorp ELISA plates (Nalge Nunc) at 4°C overnight. After washing and a 30-min incubation with 5% FCS in PBS at room temperature, 50 µl of sample or standard dilution of murine rIL-4 (PharMingen) was added. Plates were incubated overnight at 4°C, and biotinylated anti-IL-4 (1BVD6-24G2, 0.5 µg/ml) (PharMingen) was added for 3 h at room temperature following washing. Plates were washed and streptavidin/peroxidase (Sigma) was added for 30 min. Wells were developed with 3,3',5,5'-tetramethyl-benzidine substrate (Sigma), and the reaction was stopped with 1 N HCl. Plates were read at 450 nm on a microtiter plate reader (Wallac, Gaithersburg, MD). Cytokine concentrations (pg/ml) were determined by comparing the OD of samples to the standard curve.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Transduction is a process following infection by
retroviral vectors that allows chromosomal integration of the
transferred genes and provides stable cellular gene expression. In
experiments described below, we used the retroviral vector, pGCIRES (an
MMLV-based vector) engineered to contain a multiple cloning site for
incorporation of genes expressing "regulatory" proteins and a
downstream selectable marker, GFP (Fig. 1
A). The IRES sequence
contains a portion of the 5' untranslated region from the EMCV to allow
cap-independent translation (29, 30). Incorporation of the
IRES sequence allowed the formation of bicistronic RNA transcripts from
a single promoter for expression of both the upstream gene, as well as
the downstream reporter gene. The strong promoter and enhancer domains
in the 5' LTR of this vector resulted in constitutive production of the
bicistronic viral RNA transcript. As regulatory proteins, the murine
Th2 cytokine IL-4 or the Ig fusion protein for the murine sTNFR were
subcloned into the pGCIRES vector (pGCIRES.sTNFR and pGCIRES.IL4) (Fig. 1A). The pGCIRES-based retroviral vectors were used for
subsequent transduction experiments.
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Previously, reduced GFP emission levels associated with low
integration frequency in murine retroviral-mediated gene transfer
experiments has made GFP selection of transduced murine cells extremely
difficult (31). In these studies, we have utilized an
enhanced GFP marker gene with site-specific mutations in the native
jellyfish gene (using human codon preferences) that augmented
fluorescence when expressed in mammalian cells. Recombinant retrovirus
prepared from retroviral vectors that contained the wild-type GFP
(pWTGFP) or the humanized GFP variant (pGCIRES) were used to infect
murine 3T3 cells. Infected cells were analyzed by flow cytometry for
GFP expression as a measure of cell transduction. The mean channel of
fluorescence for GFP emission in murine cells using the pGCIRES
recombinant retrovirus was approximately three orders of magnitude
higher than background levels, and two orders of magnitude higher than
the fluorescence emission using wild-type GFP (pWTGFP) recombinant
retrovirus (Fig. 1
B).
Retroviral transduction of TCR transgenic autoantigen-specific CD4+ T lymphocytes
Because it has been well established that retroviruses
productively infect only dividing cells, we asked whether retroviral
infection could transduce Ag-activated CD4+ T
cells transgenic for a TCR that recognized a known Ag. As a model
system, we used B10.PL mice transgenic for a TCR reactive with MBP
(NAc1–11). CD4+ T cells
from MBP-TCR transgenic mice are >95% Ag specific
(Vß8.2+) (25). To assess
retroviral transduction in naive Ag-reactive CD4+
T cells, splenocytes from either nontransgenic PL/J or MBP-TCR
transgenic mice were stimulated in vitro with Ag (MBP peptide
NAc1–11) or with PMA and ionomycin, then exposed
to pGCIRES recombinant retrovirus (Fig. 2A). Polyclonal activation
using PMA and ionomycin stimulation resulted in nonspecific
transduction of both CD4 positive and CD4 negative lymphocytes, and
included CD8+ T cells,
B220+ B cells and GR1+
granulocytes (data not shown). However, stimulation with MBP limited
transduction to the CD4+ T lymphocytes (51.8%).
Subsequent analysis of Ag-stimulated cultures using a
transgene-specific TCR marker (anti-Vß8.2), demonstrated that the
transduced, GFP expressing cell population was limited to the TCR
Vß8.2 MBP-specific CD4+ T lymphocytes (48.8%)
and not due to the transduction of non-T cell
CD4+ populations (CD11b+,
<2%; and NK1.1+, <1%) (data not shown). In
contrast, control populations of nontransgenic PL/J splenocytes
stimulated in vitro with the MBP peptide were not demonstrably
transduced (0.4%).
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B represent cultures set up in parallel with a
constant number of PL/J splenocytes and increasing numbers of MBP TCR
transgenic splenocytes. Cultures were then stimulated in vitro with
specific Ag (MBP) or with polyclonal activation (PMA and ionomycin) for
24 h and exposed to pGCIRES retrovirus. Because the transduction
efficiency in this representative experiment of MBP-specific
CD4+ T cells was 
50% (Fig. 2A),
the expected frequency of transduced CD4+ T cells
represented approximately half the number of MBP-specific transgenic
splenocyte cells seeded into the mixed population (Table I). As anticipated for the Ag-activated
populations, the frequency of transduced CD4+ T
cells would represent approximately half of the Ag responsive
transgenic TCR-bearing MBP-specific splenocytes present in the total
population (Fig. 2B). Analyses using the transgene-specific
TCR marker (Vß8.2) confirmed that the GFP-expressing cell populations
were limited to the Vß8.2 MBP-reactive CD4+ T
lymphocytes (data not shown). Conversely, populations stimulated by
polyclonal activation showed no selective transduction of Ag-specific
(Vß8.2) CD4+ T cells. Moreover, polyclonal
activation resulted only in the transient transduction of murine
splenocytes due to activation-induced cell death exhibited by PMA and
ionomycin-stimulated populations shortly following the transduction
analysis (data not shown). Importantly, these data demonstrated that
stimulation of murine T cells with specific Ag resulted in stable
retroviral transduction of only the Ag-reactive T cells. Subsequent
experiments in our laboratory confirmed that CD4+
T cells, stimulated with specific Ag and targeted for transduction
using pGCIRES retrovirus, could be efficiently expanded in vitro by
restimulation at 7- to 10-day intervals with peptide and irradiated APC
(data not shown). In experiments conducted with four rounds of
restimulation, aliquots of transduced MBP-reactive
CD4+ T cell lines were analyzed by flow cytometry
for GFP reporter expression and assayed by ELISA for expression of
cloned regulatory products and confirmed that retroviral-introduced
transgene expression was stable for the duration of the experiments
(>40 days) with peak transgene expression occurring 48–72 h following
each round of restimulation. Moreover, the transduction of retroviral
elements and the expression of recombinant transgenes did not alter the
cytokine profile or the cell surface phenotype of the activated
CD4+ T cell (G. L. Costa and J. M.
Benson, unpublished results).
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Due to the therapeutic potential of tissue-specific expression of
regulatory proteins in animal models of autoimmunity, pGCIRES
retrovirus expressing the regulatory cytokine, IL-4, was used to infect
splenocytes from MBP TCR transgenic mice. Confirmation of augmented
fluorescence using the enhanced GFP variant in murine
CD4+ T cells was evidenced by a 20-fold higher
GFP emission fluorescence, over background levels, in
pGCIRES-transduced MBP TCR transgenic CD4+ T
cells. In contrast, the wild-type GFP recombinant retrovirus
exhibited only a 2-fold increase in fluorescence emission over
background levels (data not shown). Several independent experiments
evaluating the infection of splenocytes from MBP TCR transgenic mice
with either pGCIRES or pGCIRES.IL-4 retrovirus demonstrated
transduction efficiencies as high as 80% (data not shown). Studies to
evaluate the ability of the IRES element to allow GFP reporter
expression to be used as an indicator of upstream gene expression were
conducted using the pGCIRES.IL-4 recombinant retrovirus. Splenocytes
from MBP TCR transgenic mice were stimulated in vitro with MBP peptide,
and infected with pGCIRES, pGCIRES.IL-4, or mock recombinant
retrovirus. Consistent with previous analyses, only the Ag-specific
CD4+ T cells from Ag-activated splenocyte
cultures were transduced (Fig. 3A). Transduction
efficiencies of the CD4+ T cell populations
infected with pGCIRES and pGCIRES.IL-4 recombinant retrovirus
were 63.5% and 59.1%, respectively (Fig. 3B). The
transduced CD4+ TCR transgenic populations were
then sorted for low, medium, or high levels of GFP reporter expression.
Sorted populations were rested in culture and on day 7 postsort cells
were restimulated with MBP peptide, and supernatants were collected for
analysis of IL-4 expression by ELISA. Supernatants collected from the
TCR transgenic CD4+ T cells transduced with
vector alone (pGCIRES) or TCR transgenic CD4+ T
cells cultured in the absence of recombinant vector (mock) showed no
detectable levels of IL-4 protein (Fig. 3C). However,
protein levels of IL-4 in pGCIRES.IL-4 low, medium, and high
populations directly correlated with GFP expression levels (Fig. 3, C and D). Subsequent analysis of low, medium, and
high expressing GFP populations revealed that the GFP reporter
phenotype maintained the defined level of fluorescence over time
(data not shown). Importantly, these studies demonstrated a linear
relationship between GFP expression and upstream gene expression in
pGCIRES.IL-4-transduced CD4+ T cells. Thus, the
pGCIRES retroviral construct allowed GFP reporter gene
expression to be used as a direct measure of the upstream gene
expression in transduced murine CD4+ T cells.
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To demonstrate that rare populations of "conventional" Ag-specific CD4+ T cells in primed lymph nodes could be targeted for retroviral transduction, we used DBA/2 mice primed in vivo with SWM peptide 110–121. At 8–10 days postimmunization, draining lymph node and spleen cells were harvested and cultured in vitro with or without the immunizing Ag (SWM). Aliquots of cells were removed from culture each day for 7 days and analyzed for proliferation. Peak proliferation, as demonstrated by incorporation of tritiated thymidine, occurred in spleen and lymph node cultures on days 3 and 4 of stimulation in vitro (data not shown).
In an attempt to capture Ag-reactive cells from primed mice, draining
inguinal lymph node cells from SWM-immunized mice were activated in
vitro with immunizing peptide, infected with retrovirus, and
administered exogenous IL-2 to induce T cell cycling to facilitate
retroviral integration following Ag stimulation in vitro. As a control,
spleen cells from the immunized mice were infected with retrovirus in
parallel and represented a nondraining lymphoid population that would
be expected to contain lower frequencies of Ag-reactive cells.
Following infection with pGCIRES recombinant retrovirus, lymph node and
spleen cell populations were analyzed for transduction using flow
cytometry for the evaluation of CD4+ T cells
expressing the GFP reporter protein. In a representative experiment,
23% of the total Ag-activated lymph node population, which represents
36% of total CD4+ T cells, was transduced
following a 16-h exposure to pGCIRES recombinant retrovirus as
demonstrated by GFP reporter expression (Fig. 4). However, only 2% of the Ag-activated
cells from the spleen cell population (7% of total
CD4+ T cells) were transduced. The transduced
non-CD4+ T cells represent cycling B cells in response to
exogenously added IL-2 (data not shown). Cells from SWM-stimulated
cultures transduced with retroviral supernatant lacking vector DNA
(mock) demonstrated only background levels of GFP expression (<1%)
(Fig. 4). Lymph node or spleen cells from primed mice exposed to
pGCIRES recombinant retrovirus and exogenous IL-2, in the absence
of the immunizing Ag in vitro, were not infected above background
levels (data not shown). Using this model of retroviral infection, we
demonstrated transduction efficiencies that were higher than the
expected number of Ag-specific CD4+ T cells
postulated to exist in draining lymphoid organs (1:5) by limiting
dilution studies performed in other animal model systems
(32). In subsequent experiments not shown here, it was
revealed that the SWM-reactive Ag-specific T cell population
(Vß8+) accounted for 20% of the transduced
CD4+ T cell population with the remainder of the
transduced population most likely representative of
"adjuvant-activated," possibly purified protein derivative
(PPD)-reactive T cells activated in vivo during the priming
immunization with CFA and captured in vitro by the addition of
IL-2 during retroviral transduction. Most importantly, these studies
demonstrated that retroviral infection can be used to target rare
populations of conventional nontransgenic, Ag-reactive
CD4+ T cells following a single exposure to
pGCIRES recombinant retrovirus.
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Previous studies from our laboratory have demonstrated that
Ag-specific, proliferating T cells exhibit a
CD4high phenotype (28). We
hypothesized that transduction of Ag-specific T cells should
preferentially correlate with the CD4high
phenotype. Initial evidence for the preferential infection of
CD4high T cells was evident upon observation of
transduced populations following exposure to recombinant retrovirus. As
represented in Fig. 4, lymph node populations stained with Abs to CD4
demonstrate two distinct populations, normal and high, with the
CD4high-staining cells exhibiting preferential
expression of GFP. Therefore, to test the hypothesis that Ag-specific,
proliferating T cells exhibiting a CD4high
phenotype are preferentially targeted for retroviral transduction,
groups of DBA/2 mice were immunized with SWM peptide and draining lymph
node cells were harvested and cultured with immunizing Ag in vitro.
Following 3 days of in vitro stimulation, cultures were stained with
Abs to CD4 and sorted using flow cytometry into two distinct groups,
CD4normal and CD4high (Fig. 5A). Following cell sorting,
CD4normal and CD4high
subpopulations were stained with PI to capture the cell cycle profile
of sorted populations before retroviral infection. DNA content was
analyzed by flow cytometry, and the percentages of
CD4+ T cells that were nondividing
(G0/G1), undergoing DNA
synthesis (S) or dividing (G2/M) were determined
(Fig. 5B). Only the CD4high
subpopulation contained actively dividing cells, represented by a
population in G2/M. In contrast, the
CD4normal subpopulation contained no actively
dividing cells and the majority of cells were in
G0/G1. Sorted populations
were then exposed to pGCIRES.sTNFR recombinant retrovirus for a
decreased infection time of 2 h to allow for the transduction of
populations with known cell cycle profiles. Transduction efficiencies
of the sorted CD4+ populations were determined by
expression of GFP using flow cytometry (Fig. 5C).
Experiments demonstrated in Fig. 5 revealed that only the
CD4high population contained transduced cells and
that the number of transduced T cells (17.3%) correlated with the
frequency of cells in G2/M (21%). Conversely,
cells from the CD4normal subpopulation contained
no transduced T cells (<0.5%) and no detectable proliferating cells
(0%). Peak transduction efficiencies were routinely observed when
CD4high T cells from immunized mice were exposed
to recombinant retrovirus 3–4 days following Ag stimulation in vitro
which correlated with peak T cell proliferation profiles exhibited in
this model system (data not shown). Transduction efficiencies of
CD4normal T cells were negligible at all time
periods tested (days 1–7 post-Ag stimulation) (data not shown). These
experiments demonstrated that the CD4high
phenotype can be used as a marker of Ag reactivity in T cells and that
transduction was efficient, but limited to
CD4high T cells that were in transit through the
M phase at the time of infection. Thus, proliferating, Ag-specific
cells can be targeted by infection with retrovirus and isolated by
expression of the GFP marker protein.
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The relationship between transduction efficiency and cells in
G2/M was consistent in replicate experiments and
confirms our previous report which demonstrated that the
CD4high population contained all of the
Ag-reactive CD4+ T cells (28). To
further demonstrate the influence of cell cycle on Ag-specific
CD4+ T cell transduction, SWM-specific
CD4high T cells were sorted from lymph node cell
cultures following 3 days of stimulation in vitro and grown overnight
in the presence of IL-2 to induce T cell cycling.
CD4high T cells were then stained with a Hoechst
vital DNA stain (Hoechst 33342) and analyzed using flow cytometry for
cell cycle distribution
(G0/G1,
S+G2/M, and M subpopulations) according to DNA
content (boxed regions, Fig. 6A). As expected, the addition
of IL-2 increased the number of cycling cells as evidenced by a
striking increase in the number of cells cycling through M phase (Fig. 5B vs Fig. 6A). Viable cell cycle populations
were then separated into fractions enriched for cells in
G0/G1,
S+G2/M, or M, and exposed to recombinant
retrovirus. For the G0/G1
and S+G2/M fractions, cells were sorted from the
central third of their respective staining gates, whereas cells sorted
for the M fraction originated from the distal third of the staining
gate (shaded regions in Fig. 6A). As expected, using GFP
expression as a measure of transduction, we found preferential (almost
exclusive) retroviral infection of cells fractionated from the M phase
subpopulation (73%). None of the cells from the
G0/G1 phase were
productively infected (<0.5%), and only a small percentage of cells
from the S+G2/M phase were infected (<4%) and
most likely represented contaminating G2/M cells
within the sorted fraction (Fig. 6B). Taken together, these
analyses confirmed that efficient retroviral-mediated transduction of
CD4high T cells occurred only when cells were
actively dividing and in the M phase of the cell cycle.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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One drawback to retroviral-mediated gene therapy has been the inability to transduce nondividing cells by retroviruses. However, autoantigen-reactive CD4+ T cells proliferate and therefore provide a population suitable for retroviral transduction and for the potential targeted delivery of regulatory (or therapeutic) proteins. In most cell-mediated autoimmune diseases, inflammatory Th1 type T cells reside in the inflamed lesions. To control this inflammation, it is necessary to obtain T cells that can traffic to the lesions and regulate the inflammatory cytokines. To achieve this goal, a robust system of retroviral transduction that would transduce autoantigen-responsive CD4+ T cells was required.
It has been established that oncoretroviruses have the ability to infect dividing cells; therefore, polyclonal activation is generally used as a means to induce proliferation. Data presented here demonstrate that polyclonal activation, before retroviral infection, allowed only transient and nonspecific transduction of multiple murine lymphoid cell lineages, including CD4+ T cells. However, for applications of T cell-based gene therapy, it was only necessary for Ag-specific CD4+ T cell populations to be targeted for retroviral transduction and that transduction of the targeted CD4+ T cells be stable over time. As a model system, we first used TCR transgenic mice containing a population of CD4+ T cells specific for the MBP autoantigen (NAc1–11). CD4+ T cells isolated from MBP-specific TCR transgenic mice allowed us to evaluate the ability of our retroviral vectors to specifically target Ag-activated CD4+ T cells in vitro.
Previous barriers in the application of gene therapy to mouse models of autoimmune diseases have included low proviral integration frequency in immune cells, proviral promoter shutdown, and inadequate isolation and expansion of transduced immunoregulatory cells. Studies presented here demonstrated that Ag-activated CD4+ T cells could be transduced by pGCIRES recombinant retrovirus after stimulation in vitro with specific peptide. Using MBP-TCR transgenic mice, transduced Ag-specific CD4+ T cells exhibited robust infection frequencies (>50%) and stable transgene expression. Transduction using pGCIRES recombinant retrovirus was used to generate MBP-reactive CD4+ T cell lines that demonstrated stable expression of transgenes following four rounds of restimulation with Ag and APC (>40 days). Transgene expression appeared to follow the activation profile of the T cell as evidenced by peak transgene expression 2–3 days following each round of restimulation. Additionally, retroviral transduction of recombinant transgenes did not alter the cytokine profile or the cell surface phenotype of resting and/or activated CD4+ T cells (G. L. Costa and J. M. Benson, unpublished results).
An inherent problem of retroviral transduction is that the transduced populations are heterogenous and contain random integration(s) of provirus and thus relatively random expression of the integrated gene product. For use in gene therapy, a regulated and quantifiable gene product is desired. We solved one of these issues by demonstrating that expression of the marker protein, GFP, exhibited a linear correlation with upstream gene expression. Thus, using this system, it was possible to select the quantity of "regulatory" protein secreted by the transduced T cells based upon GFP expression. Previous retroviral-mediated delivery systems have used antibiotic- or drug-resistance to select transduced cells (34). However, a drawback of drug selection has been the inability to select populations of transduced cells for multiple (or certainly optimal) therapeutic doses of regulatory proteins. Using pGCIRES recombinant retrovirus for transduction of Ag-specific CD4+ T cells will allow expression of the marker protein, GFP, to select for multiple dose or optimal drug delivery.
Ag-specific CD4+ T cells normally exist at very
low frequency in naive as well as in memory T cell pools. Limiting
dilution studies have established the frequency of Ag-specific T cells
in a naive animal’s lymph node at 1 in 50,000–300,000 (35, 36). Only upon restimulation with specific Ag in vivo do
CD4+ T cells undergo extensive expansion that
results in an increase in cell number of 150-fold. In model systems
defining T cell reactivity in actively primed and rechallenged mice, it
has been found that frequencies of Ag-reactive T cells are 1 in 5000
(37). Application of retroviral transduction of
autoantigen-reactive CD4+ T cells in gene therapy
of autoimmunity must include systems capable of targeting these rare
populations of Ag-activated T cells.
As a model system for targeting Ag responsive cells in vivo, we used
the DBA/2 CD4+ T cell response to SWM
(28). In this system, it was previously demonstrated that
the T cells with up-regulated cell surface expression of CD4
(CD4high) contained the proliferating,
Ag-reactive T cells. Upon retroviral infection of Ag-reactivated bulk
cultures, containing either lymph node or spleen cells from
SWM-immunized mice, we found that the majority of transduced cells were
CD4high T cells (Fig. 4). To confirm our
preliminary observations that only CD4high T
cells (representing the Ag-reactive T cells) were transduced following
retroviral infection, we sorted murine T cell populations on levels of
CD4 surface expression, and demonstrated that retroviral infection of
an enriched population of CD4high T cells
resulted in enhanced transduction efficiency. The
CD4normal cells (non-Ag reactive) were not
transduced above background.
Because retroviruses integrate the chromosomal DNA of actively
dividing cells, we used cell cycle analysis to demonstrate a
correlation between CD4+ T cells traversing
mitosis and retroviral transduction. Only the
CD4high T cell population, containing the
Ag-activated CD4+ T cells cycling through
G2/M and M, were transduced. We were able to
enhance cell cycling and increase cell transduction by the addition of
IL-2 to the Ag reactivated T cell cultures. Ag-stimulated
CD4high T cells, supplemented with exogenous IL-2
for 24 h before cell cycle analyses, exhibited a dramatic
enrichment of cells cycling through mitosis. Although IL-2 was
necessary for the optimization of transduction in Ag-stimulated
CD4+ T cells, exogenously added IL-2, in the
absence of specific Ag, did not support efficient transduction of
CD4+ T cells (Fig. 3). The application of
retroviral transduction coupled with exogenously added IL-2, which
serves to both facilitate retroviral transduction and to expand target
Ag-activated T cells, should prove beneficial when attempting to
isolate rare populations of autoreactive cells with unknown Ag(s)
specificity, as in animal models for human diseases such as multiple
sclerosis, diabetes, and rheumatoid arthritis.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. C. Garrison Fathman, Department of Medicine, Division of Immunology and Rheumatology, Room S-021, Stanford University School of Medicine, Stanford, CA 94305-5111. E-mail addresses:
3 Abbreviations used in this paper: GFP, green fluorescent protein; MMLV, Moloney murine leukemia virus; LTR, long terminal repeat; , psi packaging signal; EMCV, encephalomyocarditis virus; IRES, internal ribosome entry site; MBP, myelin basic protein; SWM, sperm whale myoglobin; sTNFR, soluble TNF receptor; PI, propidium iodide.
Received for publication October 25, 1999. Accepted for publication January 27, 2000.
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