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Journal of Virology, January 2001, p. 799-808, Vol. 75, No. 2
0022-538X/01/$04.00+0 DOI: 10.1128/JVI.75.2.799-808.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Nonmyeloablative Immunosuppressive Regimen Prolongs
In Vivo Persistence of Gene-Modified Autologous T Cells in a Nonhuman
Primate Model
Carolina
Berger,1
Meei-Li
Huang,1
Michael
Gough,2
Philip D.
Greenberg,1,3,4
Stanley R.
Riddell,1,2 and
Hans-Peter
Kiem1,2,3,*
Fred Hutchinson Cancer Research Center,
Seattle, Washington 98109,1 Departments
of Medicine3 and
Immunology,4 University of Washington,
and the University of Washington Regional Primate Research
Center,2 Seattle, Washington 98195
Received 22 August 2000/Accepted 18 October 2000
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ABSTRACT |
The in vivo persistence of gene-modified cells can be limited by
host immune responses to transgene-encoded proteins. In this study we
evaluated in a nonhuman primate model whether the administration of a
nonmyeloablative regimen consisting of low-dose total-body irradiation
with 200 cGy followed by immunosuppression with mycophenolate mofetil
and cyclosporin A for 28 and 35 days, respectively, could be used to
facilitate persistence of autologous gene-modified T cells when a
transgene-specific immune response had already been established or to
induce long-lasting tolerance in unprimed recipients. Two macaques
(Macaca nemestrina) received infusions of T cells
transduced to express either the enhanced green fluorescent protein and
neomycin phosphotransferase genes or the hygromycin phosphotransferase
and herpes simplex virus thymidine kinase genes. In the absence of
immunosuppression, both macaques developed potent class I major
histocompatibility complex-restricted CD8+ cytotoxic
T-lymphocyte (CTL) responses that rapidly eliminated the gene-modified
T cells and that persisted long term as memory CTL. Treatment with the
nonmyeloablative regimen failed to abrogate preexisting memory CTL
responses but interfered with the induction of transgene-specific CTL
and facilitated in vivo persistence of gene-modified cells in an
unprimed host. However, sustained tolerance to gene-modified T cells
was not achieved with this regimen, indicating that further
modifications will be required to permit sustained persistence of
gene-modified T cells.
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INTRODUCTION |
Genetically modified cells are being
evaluated in studies for the correction of genetic diseases and to
treat acquired diseases such as cancer and AIDS (2, 10, 25,
30). A potential obstacle to the in vivo persistence of these
cells is the development of host immune responses to novel
transgene-encoded proteins (38). Clinical trials and
studies in immunocompetent animals demonstrated that cells modified
with retroviral vectors to express a therapeutic gene and/or a
selectable marker transgene such as the neomycin phosphotransferase
(neo), hygromycin phosphotransferase (Hy), herpes simplex
virus thymidine kinase (TK), or enhanced green fluorescent protein
(EGFP) gene can elicit potent host immune responses (5, 28, 38,
47, 49). The immune mechanisms responsible for eliminating
genetically altered cells included both CD8+ cytotoxic
T-lymphocyte (CTL) responses specific for peptide fragments derived
from intracellular proteins presented in association with class I major
histocompatibility complex (MHC) molecules and/or antibody responses to
transgene products expressed at the cell surface.
Several strategies have been investigated to overcome the obstacle of
immune recognition. The coexpression of viral immune evasion genes,
which have been identified to selectively interfere with class I MHC
presentation of target antigens, can prevent CTL-mediated lysis of
cells constitutively expressing transgene proteins in vitro (3,
36, 42). However, this strategy is restricted to intracellularly
expressed proteins and might be limited in vivo by recognition of
modified cells by natural killer cells, which preferentially eliminate
cells lacking class I MHC molecules on the cell surface (19,
26).
Immunosuppressive regimens commonly applied following solid organ
transplantation or hematopoietic stem cell transplantation (HSCT) have
also been explored to circumvent host immune responses to vector- or
transgene-encoded proteins. Long-term administration of cyclosporine A
(CSP) alone or with methotrexate failed to abrogate host immune
responses (11, 15, 28). Although a combination of CSP and
cyclophosphamide prolonged in vivo gene expression after
adenovirus-mediated gene transfer, secondary gene delivery was not
tested and long-term immunosuppression with this approach is limited by
the risk of infectious complications and other toxicities (11).
Recent studies by Storb et al. demonstrated that a low (200-cGy) dose
of total-body irradiation (TBI) followed by transient postgrafting
immunosuppression with mycophenolate mofetil (MMF) and CSP for 28 and
35 days, respectively, can induce stable mixed chimerism following
allogeneic HSCT in a canine model and in humans (45, 46).
This regimen is nonmyeloablative and causes only transient reduction in
neutrophil and platelet counts. Moreover, recent studies using a
nonmyeloablative regimen in the setting of autologous HSCT demonstrated
sustained in vivo persistence of stem cells modified to express a
marker gene, suggesting that this approach may circumvent the
immunogenicity of transgene-encoded proteins (18, 41). In
many settings, differentiated somatic cells rather than stem cells are
used as vehicles for gene delivery. T cells can be efficiently modified
by gene transfer, and studies in which a normal adenosine deaminase
gene was introduced into autologous T cells demonstrated that these
cells can be long-lived in vivo following transfer in immunodeficient
hosts (4, 6). Unfortunately, the use of gene-modified T
cells in immunocompetent hosts has been complicated by immune responses
to transgene products (28, 38, 47). Therefore, we sought
to determine in a nonhuman primate model (Macaca nemestrina)
whether nonmyeloablative conditioning with low-dose TBI (200 cGy), MMF,
and CSP could facilitate the persistence of gene-modified T cells in
settings in which a transgene-specific memory T-cell response was
already established or in which the transgene-encoded protein
represented a neoantigen. The regimen failed to overcome an established
transgene-specific memory CTL response but did interfere with the
induction of a primary transgene-specific CD8+ CTL response
and markedly prolonged the in vivo persistence of gene-modified T cells
in an unprimed host.
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MATERIALS AND METHODS |
Retroviral vectors.
The retroviral vector pLGSN was
constructed by ligating the EcoRI-NotI (Klenow
filled) 767-bp fragment of pEGFP-1 into EcoRI- and
HpaI-digested pLXSN (32). Vector stocks were
made by using PG13 retrovirus-producing packaging cells
(31). The LN (PG13) vector carrying the neo
gene alone has been described elsewhere (32). The
retroviral vector MSCVEGFP (PG13), carrying the EGFP gene, was provided
by R. G. Hawley (University of Toronto, Toronto, Ontario, Canada).
The retroviral vector HyTK (PA317), carrying the Hy and the herpes
simplex virus TK genes as a single fusion gene, was provided by
Targeted Genetics (Seattle, Wash.) (27). The PG13- and
PA317 packaging cell lines were grown in Dulbecco's modified Eagle
medium supplemented with 10% fetal bovine serum (HyClone Laboratories,
Logan, Utah).
Transduction and expansion of M. nemestrina
lymphocytes.
Peripheral blood mononuclear cells (PBMC) were
separated by Ficoll-Hypaque 1077 density gradient centrifugation and
then cultured in RPMI 1640 medium supplemented with 25 mM HEPES, 11%
human AB serum (Gemini Bio-Products, Calabasas, Calif.), 25 µM
2-mercaptoethanol (Sigma Chemical Co., St. Louis, Mo.), 4 mM
L-glutamine (GIBCO-BRL, Gaithersburg, Md.), and 2%
purified human interleukin-2 (IL-2; Advanced Biotechnologies
Incorporation, Columbia, Md.). Prior to transduction, cells were
stimulated for 36 to 48 h with the immobilized anti-CD3 (SP34; 10 to 20 ng/ml; PharMingen, San Diego, Calif.), and anti-CD28 (9.3; 1 µg/ml; P. Martin, Fred Hutchinson Cancer Research Center, Seattle,
Wash.) monoclonal antibodies (MAbs) and then placed into six-well
plates that had been seeded 12 h earlier with 106
-irradiated (2,500 rad) packaging cells producing either the LGSN,
LN, or HyTK retrovirus vector. After 48 h of coculture in the
presence of protamine sulfate (8 µg/ml), nonadherent cells were
collected and replated in fresh medium. For selection of transduced
cells, either Geneticin (G418 sulfate; 1.0 mg/ml; GIBCO-BRL) or
hygromycin B (0.2 mg/ml; Boehringer Mannheim, Indianapolis, Ind.) was
added after 24 h. T cells were then expanded using anti-CD3 (10 ng/ml) and anti-CD28 (1 µg/ml) MAbs, in the presence of
-irradiated human PBMC and herpesvirus papio-transformed macaque
B-lymphoblastoid cell lines (B-LCL), and IL-2 as described previously
(39). On day 13, cells from these cultures were
cryopreserved in aliquots which could be thawed subsequently for
further expansion and reinfusion.
To expand T cells for adoptive transfer, aliquots of cells transduced
to express either the EGFP and neo genes or the HyTK gene
were stimulated in 25-cm2 flasks. On day 13, T cells were
harvested, washed three times with Dulbecco's phosphate-buffered
saline, and resuspended in 50 ml of isotonic saline solution containing
2% autologous serum for infusion. Prior to infusion, cells were tested
by flow cytometry for expression of EGFP, CD3, CD4, and CD8 and by PCR
for the presence of the transgene.
Generation and transduction of B-LCL.
B-LCL were established
by infecting PBMC with fresh supernatant from S394-1X1055 cells
containing herpesvirus papio as described previously (20).
B-LCL were transduced with the LGSN, LN, MSVEGFP, or HyTK retroviral
vector by cocultivation as described above and either selected with
G418 (1 mg/ml) or hygromycin B (0.2 mg/ml) or sorted for expression of
EGFP on a Ventage instrument (Becton Dickinson, Mountain View, Calif.).
Animals and experimental design.
Healthy adult pig-tailed
macaques (M. nemestrina) were housed at the University of
Washington Regional Primate Research Center under American Association
for Accreditation of Laboratory Animal Care-approved conditions.
Protocols were approved by the Institutional Review Board and Animal
Care and Use Committee. All procedures and blood draws were performed
as described elsewhere (24, 33).
A schematic diagram of the experimental design is shown in Fig.
1. Previous clinical trials indicated
that a gene-modified
T-cell dose of 10
9/m
2
resulted in an easily detectable frequency of transferred cells
in
peripheral blood (
8,
38). Thus, this cell dose was chosen
for each infusion in this study. For immunosuppression, a single
dose
of TBI (200 cGy) was given from opposing
60Co sources (7 cGy/min) prior to the T-cell infusion. CSP (1.5
mg/kg intravenously
[i.v.] twice a day [b.i.d.]; Sandoz Pharmaceuticals
Corporation,
East Hanover, N.J.) was begun 3 days before TBI was
administered to
ensure a sufficient drug level, and MMF (10 mg/kg
i.v. b.i.d.; donated
by Roche Laboratories Inc., Nutley, N.J.)
was started on the day T
cells were transferred. MMF and CSP were
continued for 4 and 5 weeks,
respectively (
46). Drug levels
were monitored, and doses
were adjusted as needed. Standard supportive
care was provided
(
24). A lymph node biopsy was performed 3
days after the
last cell dose.
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FIG. 1.
Schematic diagram of the experimental design. Each arrow
indicates an infusion of 109 autologous gene-modified T
cells/m2. For immunosuppression, macaque 90152 was given a
nonmyeloablative dose (200 cGy) of TBI prior to the infusion of both
EGFP/neo- and HyTK-modified T cells (day 140). CSP (1.5 mg/kg b.i.d. i.v.) was administered on days 137 to 175, and MMF (10 to
15 mg/kg b.i.d. i.v.) was given on days 140 to 168.
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Flow cytometric analysis and cell sorting.
PBMC and T cells
were analyzed for expression of CD3 by indirect immunofluorescence
using the murine immunoglobulin (Ig) G3 MAb SP34 (PharMingen) as
primary antibody followed by fluorescein isothiocyanate-conjugated or
phycoerythrin-conjugated goat-anti-mouse IgG MAb (Tago Immunologicals,
Camarillo, Calif.) as secondary antibody. Fluorescein isothiocyanate-
or phycoerythrin-conjugated antibodies used for immunophenotyping
included anti-CD4 (Leu 3a), anti-CD8 (Leu 2a), anti-CD16 (Leu 11a), and
anti-CD20 (Leu 16) MAbs and isotype-matched irrelevant control
antibodies (Becton Dickinson). Transferred EGFP-expressing T cells were
enumerated in PBMC by analyzing >100,000 gated events for each
analysis. PBMC from control animals were used to define negative
fluorescence gates, and dead cells were excluded based on staining with
propidium iodide (1 µg/ml; Sigma). Fluorescence analysis was
performed on a FACScan flow cytometer (Becton Dickinson), and data were
analyzed using CellQuest software.
Lymphoproliferative assay.
PBMC (105/well) were
plated in triplicate and stimulated with concavalin A (ConA; 10 µg/ml; Sigma) or anti-CD3 and anti-CD28 MAbs as described above and
incubated for 96 h at 37°C. For mixed lymphocyte reactions, an
equal number of -irradiated (3,500 rad) allogeneic PBMC was added.
Cells were pulsed with [3H]thymidine (2.5 µCi/well; NEN
Products, Boston, Mass.) 16 h before harvest. In some experiments,
cells were incubated in the presence of CSP (0.01 to 10 µg/ml) or
mycophenolic acid (0.01 to 10 µM; Sigma), the pharmacologically
active compound of the prodrug form MMF. The stimulation index was
calculated as described elsewhere (8).
Cytotoxicity assays.
Cytotoxic responses specific to EGFP,
neo, or HyTK were assessed by methods described previously
(3, 38). Briefly, PBMC obtained from the animals before
and after infusion were stimulated at a responder-to-stimulator ratio
of 2:1 twice 1 week apart with -irradiated autologous T cells
expressing either the EGFP/neo, neo, or HyTK
gene. Cells from these cultures were assayed in a chromium release
assay at various effector-to-target (E/T) ratios for specific
recognition of autologous 51Cr-labeled B-LCL or T cells,
either parental or transduced to express EGFP, EGFP/neo,
neo, or HyTK. In some experiments, CD4+ or
CD8+ T-cell subpopulations were isolated before the assay
by using MACS MS+/RS+ separation columns
(Miltenyi Biotec, Auburn, Calif.) according to the manufacturer's
directions. To evaluate class I MHC-restricted cytolytic responses,
target cells were preincubated (60 min, 4°C) with either the class I
MHC MAb W6/32 (25 µg/ml; D. Geraghty, Fred Hutchinson Cancer Research
Center) or the anti-HLA-DR MAb L243 (HB55; American Type Culture
Collection, Manassas, Va.) (dilution of 1:10 [ascites fluid]; V. Groh, Fred Hutchinson Cancer Research Center). Both W6/32 and L243
cross-react with macaque MHC (34, 35).
LDA.
CTL precursors (CTLp) specific for EGFP or HyTK were
quantified by limiting dilution analysis (LDA) of PBMC obtained before and after transfer of T cells (38). Twenty-four replicates
of PBMC were plated in 96-well round-bottomed plates at concentrations of 375 to 100,000 cells per well with 2 × 103
autologous -irradiated (3,000 rad) EGFP- or HyTK-transduced T cells
per well as stimulators and 5 × 104 autologous
-irradiated PBMC per well as feeder cells. IL-2 (2%) was added on
day 3, 6, 9, and 12. Wells were assayed on day 14 in a chromium release
assay for specific recognition of autologous B-LCL, either parental or
expressing EGFP, neo, or HyTK. CTL frequencies and
confidence intervals were determined by maximum likelihood analysis as
described elsewhere (12, 21).
Fluorescent probe PCR assay (TaqMan).
PCR amplifications and
analysis of the neo or HyTK gene were performed by using a
quantitative real-time PCR assay (TaqMan) (16). DNA (0.3 to 1 µg) was amplified in duplicate or triplicate with
neo-specific primers (5'-GGA TTG CAC GCA GGT TCT C-3' and 5'-AGA GCA GCC GAT TGT CTG TT-3') and a fluorescence-tagged probe (5'-FAM-TGC CCA GTC ATA GCC GAA TAG CCT CTC CAT-TAMRA-3'; Synthegen, Houston, Tex.) (9). For HyTK, the specific primers 5'-TAC
ACA AAT CGC CCG CAG A-3' and 5'-AGC CTG GTC GAA CGC AGA C-3' were used
with the probe 5'-FAM-CGA CTT CTA CAC AGC CAT CGG TCC AGA-TAMRA-3'. These primers were designed using Primer Express software (Perkin-Elmer Applied Biosystems, Foster City, Calif.). Standards consisted of
dilutions of DNA extracted from a human T-cell clone transduced with a
single copy of the neo or HyTK vector (8, 38).
The limit of detection of the real-time PCR assay was ~10 copies per 106 cells. Negative controls consisted of DNA extracted
from PBMC obtained preinfusion or from control animals or water.
Samples were subjected to 50°C for 2 min and 95°C for 10 min,
followed by 42 cycles of amplification at 95°C for 25 s and
60°C for 1 min, using an ABI PRISM 7700 sequence detection system
(Perkin-Elmer).
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RESULTS |
In vivo persistence of autologous gene-modified T cells in
macaques.
Autologous T cells were transduced to express either
both the EGFP and neo genes or the HyTK gene, expanded in
vitro, and adoptively transferred twice 1 week apart to different
macaques (Fig. 1). The EGFP/neo retroviral vector was
produced in LGSN PG13 packaging cells and transduced 69.4% of the T
cells (Fig. 2A). This expression remained stable with subsequent cell
expansion, making drug selection unnecessary. A lower gene transfer
efficiency of approximately 11% was achieved following transduction of
T cells with the amphotropic PA317 HyTK packaging cells. Thus, T cells
from these cultures were selected in hygromycin B prior to expansion to
increase the fraction of cells expressing the transgene. Efficient
expansion of gene-modified macaque T cells to cell numbers of
>109 was achieved in vitro by using anti-CD3 and anti-CD28
stimulation. Phenotypic analysis of the EGFP/neo-marked T
cells prior to infusion revealed that a mean of 76.2% (range, 70.0 to
83.6%) were CD3+ CD8+ and 26.4% (range, 18.3 to 29.4%) were CD3+ CD4+. A mean of 95.6%
(range, 91.8 to 97.7%) of the HyTK+ T cells were
CD3+ CD8+ T cells, and 5.1% (range, 2.2 to
10.5%) were CD3+ CD4+.
The persistence of transferred T cells in peripheral blood was examined
by flow cytometry for the presence of EGFP-expressing
cells (Fig.
2B) and by PCR for
neo or HyTK
sequences. In the macaque
receiving EGFP/
neo-marked T cells,
5,427
neo copies per 10
6 cells were detected 30 min after the first infusion, and these
cells were slightly reduced in
frequency by days 1 and 3 (Fig.
3A).
However, only 211
neo copies per 10
6 cells were
detected by day 7, and the frequency of
neo+
cells declined rapidly over 24 h following the second cell dose
to
undetectable levels by day 3 postinfusion. Analysis of PBMC
samples by
flow cytometry for expression of EGFP confirmed the
pattern of
persistence of EGFP/
neo-marked T cells (Fig.
3A).
Transferred
EGFP-expressing cells were present for 3 days after the
first
infusion in 0.15 to 0.31% of PBMC (~1,454 to 3,142 per
10
6 cells). However, this number declined to low levels on
day 7,
and the cells were not detectable 24 h after the second
cell dose.
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FIG. 2.
(A) Successful gene transfer of M. nemestrina
T lymphocytes, demonstrated by representative flow cytometry analysis
of EGFP expression in T cells after transduction with the LGSN (PG13)
retroviral vector. Gene transfer was accomplished using an optimized
transduction protocol including anti-CD3 and anti-CD28 stimulation of T
cells. Viable cells were stained with anti-CD3 MAb and analyzed by flow
cytometry. Percentages of cells positive for both EGFP and CD3 are as
indicated. (B) Transferred EGFP-expressing T cells are detectable by
flow cytometry in vivo. PBMC from a control animal (left) and PBMC
collected from macaque 90152 at 30 min postinfusion (day 410) (right)
were analyzed for expression of both EGFP and CD3. Percentages of cells
positive for both EGFP and CD3 are as indicated.
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FIG. 3.
Analysis of PBMC for the in vivo persistence of
gene-modified T lymphocytes following in vivo priming. (A) Frequency of
EGFP/neo-modified T cells transferred to macaque 90152 in
peripheral blood. PBMC collected before and at the indicated days after
infusion were analyzed by the quantitative real-time PCR assay for the
presence of the neo sequence or by flow cytometry for
EGFP-expressing cells. For enumeration of transferred EGFP-expressing T
cells in PBMC, >100,000 viable cells were analyzed for each assay.
Negative fluorescence gates were defined using PBMC from control
animals. (B) Detection of HyTK-modified T cells transferred to macaque
97140 in circulating PBMC by the real-time PCR assay. Arrows indicate
the days of T-cell infusions.
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A similar pattern of persistence was observed in the macaque receiving
HyTK-modified T cells (Fig.
3B). Transferred cells
persisted at high
levels for 7 days after the first cell dose,
but the frequency of HyTK
copies declined >150-fold by 3 days
after the second infusion, and
HyTK-marked cells were no longer
detectable after day
14.
CD8+ CTL specific for the transgene products are
elicited after transfer of gene-modified T cells and persist as memory
cells.
To determine if the short persistence of gene-modified T
cells reflected the induction of a host immune response against
transgene-encoded proteins, PBMC were obtained from the macaques before
and at intervals after infusion and stimulated with -irradiated
autologous T cells transduced to express either EGFP/neo,
neo, or HyTK. No cytolytic activity was detected in cultures
of preinfusion PBMC, indicating the absence of primary CTL responses to
EGFP, neo, and HyTK (Fig. 4A
and D). However, in the macaque receiving LGSN-modified T cells, both
EGFP- and neo-specific CTL responses were first observed by
7 days after the first cell dose (Fig. 4B and C). In the animal receiving HyTK-expressing T cells, a HyTK-specific cytolytic response was first detectable on day 8 (Fig. 4E) and was stronger by day 10 (Fig. 4F).
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FIG. 4.
CTL responses specific for the transgene products are
elicited following transfer of gene-modified T cells. (A) Analysis of
PBMC collected from macaque 90152 prior to the infusion of
EGFP/neo-modified T cells for the presence of
EGFP/neo-specific CTL. (B and C) Analysis of PBMC collected
from macaque 90152 on day 7 following infusion of
EGFP/neo-modified T cells for the presence of EGFP-specific
CTL (B) and neo-specific CTL (C). Aliquots of PBMC were
analyzed following stimulation with autologous T cells expressing EGFP
and/or neo in a chromium release assay for recognition of
target cells, either parental ( ) or transduced to express the EGFP
( ) and neo ( ) genes alone and together ( ). (D to F)
PBMC from macaque 97140 obtained before (D) and on days 8 (E) and 10 (F) following the first cell dose were cocultured with autologous
HyTK-modified T cells and then assayed for HyTK-specific cytolytic
activity against target cells, either parental () or transduced to
express the HyTK gene ( ).
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The lytic activity of these cultures could potentially reflect several
populations of effector cells, including CD8
+ class I
MHC-restricted and/or CD4
+ class II MHC-restricted T cells
or non-MHC-restricted natural
killer cells. Positively selected
CD4
+ T cells from these cultures revealed little
EGFP/
neo- and HyTK-specific
cytolytic activity (Table
1). In contrast, enrichment of
CD8
+ T cells from these cultures augmented the cytolytic
activity,
suggesting that the lytic activity was mediated predominantly
by this subset of T cells. This was confirmed by preincubating
the
targets with anti-class I and anti-class II MHC MAbs. Treatment
of
target cells with class I MAb significantly reduced the lysis
of
autologous EGFP/
neo-expressing targets, whereas treatment
with
class II MAb had no effect (Table
2). These results indicated
that transfer
of gene-modified T cells elicited potent class I
MHC-restricted
CD8
+ CTL responses specific for epitopes derived from
transgene proteins.
We next determined whether the CTL responses persisted long term
following in vivo priming. Analysis of PBMC collected on
a biweekly
basis following priming demonstrated that strong EGFP-
and
neo-specific cytolytic reactivity persisted during the
follow-up
and was present even 10 weeks following the first cell dose
(Fig.
5A and B). Similarly, in the
macaque receiving HyTK-expressing
T cells, HyTK-specific cytolytic
reactivity was detectable in
samples of PBMC more than 12 weeks after
the first cell dose (data
not shown).
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FIG. 5.
Transferred gene-modified T cells induce potent memory
T-cell responses specific for the transgene products. (A and B)
Aliquots of PBMC from macaque 90152 were collected on day 70 prior to
the readministration of EGFP/neo-modified T cells and
cocultured with autologous T cells either expressing both EGFP and
neo (A) or neo alone (B). Cells from these
cultures were then assayed in a chromium release assay for recognition
of target cells, either parental () or expressing both the EGFP and
neo genes () or the neo () gene alone. (C)
Frequency of EGFP/neo-modified T cells readministered to
macaque 90152 in peripheral blood. PBMC collected before and at the
indicated days postinfusion were analyzed by real-time PCR for the
presence of the neo sequence as described in the text or by
flow cytometry to enumerate EGFP-expressing cells. Arrows indicate the
days of T-cell infusions.
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To evaluate whether these transgene-specific memory CTL mediated
immune elimination of gene-modified cells even after a prolonged
period, EGFP/
neo-modified T cells were readministered on
days
70 and 77 to the previously sensitized macaque 90152. The
transferred
cells were rapidly eliminated, consistent with the
establishment
of a potent memory T-cell response to the transgene
products (Fig.
5C). Moreover, LDA of samples of PBMC collected prior
and after
retransfer of T cells revealed a substantial boost of
EGFP-specific
CTLp in PBMC following reexposure to transgene-expressing
cells
from a frequency of 1 per 10,076 (95% confidence interval, 1 per
7,658 to 1 per 13,258) to 1 per 710 (95% confidence interval,
1 per
510 to 1 per 989) 6 weeks postinfusion (Table
3).
In vivo persistence of gene-modified T cells given with transient
immunosuppression.
To evaluate if administration of a transient
immunosuppressive regimen could facilitate persistence of gene-modified
T cells despite a memory CD8+ CTL response to the transgene
product or induce tolerance against a novel transgene-encoded protein,
macaque 90152, previously sensitized against EGFP and neo
but not against HyTK, received both EGFP/neo- and
HyTK-modified T cells on days 140 and 147. The T cells were infused
after a single 200-cGy dose of TBI and initiation of 28- and 35-day
courses of MMF and CSP, respectively (Fig. 1).
The EGFP/
neo-marked T cells disappeared rapidly after the
infusion with kinetics similar to those observed with the infusions
on
days 70 and 77 when EGFP/
neo-marked T cells were given
without
immunosuppression (Fig.
6A).
Thus, the immunosuppressive regimen
failed to eliminate or inhibit the
function of memory T-cell responses
to EGFP or
neo. In
contrast, the transferred HyTK-modified T cells
persisted in the
peripheral blood at high levels (>250 HyTK copies
per 10
6
cells) for more than 60 days postinfusion (Fig.
6A). The frequency
of
HyTK-modified cells then declined gradually, in part reflecting
repopulation of the peripheral lymphoid compartment after withdrawal
of
the immunosuppression. However, more than 100 copies of the
HyTK gene
per 10
6 cells were still present >100 days postinfusion.
The HyTK-marked
cells continued to decline to low levels and were
sporadically
detectable until day 371. The HyTK gene could still be
detected
if samples of PBMC collected on day 371 were cultured in the
presence
of hygromycin B (data not shown). These results suggest that
the
transient immunosuppressive regimen efficiently interfered with
the
induction of HyTK-specific CTL and permitted the prolonged
persistence
of HyTK-modified T cells in vivo.
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FIG. 6.
(A) Analysis of PBMC for the presence of both
EGFP/neo- and HyTK-modified T cells transferred with a
transient nonmyeloablative regimen to macaque 90152. The frequency of
neo or HyTK sequences was evaluated in samples of DNA
derived from PBMC collected before and on the indicated days after
infusion, using the real-time PCR assay. The frequency of transferred
EGFP-expressing T cells in the same samples of PBMC was assessed by
flow cytometry as described in the text. Arrows indicate the days of
T-cell infusions. (B) EGFP/neo- and HyTK-specific CTL
responses before and after immunosuppressive treatment. Aliquots of
PBMC obtained from macaque 90152 at the days indicated on the
horizontal axis were stimulated twice with autologous T cells
expressing either both the EGFP and neo genes or the HyTK
gene and then assayed in a chromium release assay at various E/T ratios
for recognition of target cells, either parental (white bars) or
transduced to express either both the EGFP and neo genes
(hatched bars) or the HyTK gene (black bars). The percent specific
lysis of the target cells indicated on the vertical axis is shown for
an E/T ratio of 20:1. The presence of EGFP-specific CTL was evaluated
in samples of PBMC collected on day 371 instead of day 389. The
duration of the treatment is indicated by the horizontal bar, and
arrows indicate the days of T-cell infusions.
|
|
Evaluation of CTL responses during and following
immunosuppression.
To evaluate the impact of the immunosuppressive
regimen on the preexisting CTL responses to EGFP and neo,
and the development of a primary CTL response to HyTK, samples of PBMC
were examined before and after therapy for the presence of cytolytic
activity against either EGFP, neo, or HyTK. EGFP- and
neo-specific CTL were detectable in cultures of PBMC
collected during and/or following immunosuppressive treatment (Fig.
6B). The persistence of these cytolytic effector cells was not likely
due to inadequate dosing of CSP or MMF in macaques since in vitro
exposure to levels of both CSP and mycophenolic acid comparable to
those present in vivo inhibited lymphoproliferative responses of
macaque T cells to ConA or allogeneic lymphocytes by 71.7 to 84.5% or
94.4 to 99.0%, respectively (data not shown).
In contrast, no HyTK-specific cytolytic reactivity was detected in any
of the cultures of PBMC collected until day 389 (Fig.
6B). Lymphocyte
counts of >400/µl and 800 to 1,000/µl were first
detectable after
periods of 48 and 75 days, respectively, and
lymphoproliferative
responses to ConA, anti-CD3 and anti-CD28
MAbs, and allogeneic
lymphocytes recovered after day 163 posttherapy
(data not shown). Thus,
our results indicated that the immunosuppressive
regimen blocked the
development of HyTK-specific
CTL.
The animal treated with this regimen experienced an unexpectedly
prolonged period of myelosuppression. On day 22 of the regimen,
absolute neutrophil counts declined below 500/µl and remained
at this
level until day 91, and platelet counts remained below
20,000/µl
until day 119 (study day 259). However, the animal continued
to eat and
maintained its weight, and there was no evidence of
underlying diseases
or of infectious or hemostatic
complications.
HyTK-specific CTL are elicited after secondary administration of
gene-modified T cells.
To determine if immune tolerance to the
HyTK transgene product had been induced, HyTK-modified T cells were
readministered to the animal 9 months after the immunosuppressive
treatment. At this time, lymphocyte subsets had recovered to normal
levels, and lymphoproliferative responses to mitogens such as ConA or anti-CD3 and anti-CD28 MAbs, as well as mixed lymphocyte responses, were in the normal range. Analysis of PBMC samples by PCR demonstrated that 3,316 copies of the HyTK gene per 106 cells were
detectable 30 min after the first infusion on study day 410 and were
present in a reduced frequency of 1,449 or 411 HyTK copies per
106 cells on days 1 and 3 postinfusion, respectively, but
declined to undetectable levels by day 7 (Fig.
7A). However, after the second cell dose,
the frequency of HyTK copies decreased 10-fold during 24 h and
continued to rapidly decline to <30 copies per 106 cells
on day 3. Cells became undetectable by day 7. To evaluate whether the
marked cells were sequestered in lymphoid tissues, a lymph node biopsy
was performed. PCR analysis of cell suspensions indicated the absence
of transferred cells.
View larger version (16K):
[in this window]
[in a new window]
|
FIG. 7.
HyTK-specific CTL are elicited following
readministration of HyTK-modified T cells to macaque 90152. (A) In vivo
persistence of HyTK-modified T cells. Samples of PBMC collected before
and on the indicated days after infusion were evaluated by real-time
PCR for the frequency of HyTK sequences. Arrows indicate the days of
T-cell infusions, and the star denotes an inguinal lymph node biopsy.
(B and C) CTL responses specific for target antigens derived from HyTK
in samples of PBMC collected before (study day 389) (B) and 7 days
after (study day 417) (C) infusion. PBMC were cocultured with
autologous -irradiated HyTK-expressing T cells and then assayed for
HyTK-specific cytolytic activity against target cells, either parental
() or transduced to express the HyTK gene ().
|
|
No HyTK-specific CTL response was detectable prior to readministration
of cells (Fig.
7B), but HyTK-specific cytolytic effector
T cells were
detectable on day 7 postinfusion (Fig.
7C) and persisted
during the
follow-up (data not shown). Thus, although the transient
immunosuppressive treatment delayed the initiation of a primary
immune
response to HyTK, it failed to induce permanent immune
tolerance.
| |
DISCUSSION |
A major obstacle to the in vivo persistence of gene-modified cells
is the development of a host immune response to transgene-encoded proteins (28, 38). The design of immunomodulatory
approaches to circumvent immune recognition of these cells in
immunocompetent hosts has been difficult (3, 11, 15).
Myeloablative treatment regimens could potentially be used, since host
immune responses against novel transgene-encoded proteins expressed in
gene-modified cells have not been found under these conditions in large
animal models and in patients with malignant diseases (1, 7, 13, 17, 23). However, the acute and long-term toxicities of complete myeloablative conditioning with high-dose TBI and/or chemotherapy are
undesirable in patients with nonmalignant disorders. Recent studies by
Storb et al. demonstrated that a nonmyeloablative TBI dose of 200 cGy
followed by transient immunosuppression with MMF and CSP is capable of
inducing persistent immune tolerance across MHC barriers of allogeneic
HSCT in a canine model and in humans. Therefore, we evaluated this
approach as a potential strategy to overcome immune recognition of
gene-modified T cells (45, 46).
Our results demonstrated that the transfer of autologous T cells
transduced to express either the EGFP and neo genes or the HyTK gene elicited rapid and potent class I MHC-restricted
CD8+ CTL responses specific to these transgene products.
This in vivo priming resulted in strong and persistent
transgene-specific memory T-cell responses, which were maintained
during and following a nonmyeloablative immunosuppressive regimen and
rapidly eliminated gene-modified cells readministered several months
later. These results have implications for the treatment of inherited
protein-null genetic deficiency disorders, since in this circumstance,
cells expressing the therapeutic gene product may be similarly
immunogenic. Our observations demonstrate that once a
transgene-specific CD8+ CTL response is established, its
modulation might be very difficult and immune rejection may limit
subsequent efforts to correct the defect by administering gene-modified
cells. Thus, the prevention of primary host immune responses against
transgene-encoded proteins will be essential for successful gene therapy.
In this study, the nonmyeloablative immunosuppressive regimen was
effective in preventing the induction of CTL specific for a novel
transgene product and permitted the prolonged (for >7 months)
persistence of gene-modified T cells in vivo. However, in contrast to
the nonmyeloablative allogeneic HSCT setting, in which permanent
tolerance to allogeneic hematopoietic stem cells is frequently
achieved, persistent immune tolerance to the transgene product was not
observed and reinfusion of gene-modified T cells 9 months following the
regimen led to the induction of CTL specific for the transgene product.
Several factors may explain the inability to achieve persistent
tolerance to the transgene product. First, there is evidence that the
sustained presence of the antigen is required for persistent immune
tolerance, and the shorter life span of T cells than of permanently
engrafted and self-renewing hematopoietic stem cells may have resulted
in the eventual loss of the antigen (14, 37, 40). Studies
of adenosine deaminase-deficient patients have shown that transferred
genetically corrected T cells can survive in vivo for more than 4 years
(4, 6). However, in this setting of congenital
immunodeficiency, the gene-modified T cells had a survival advantage
compared with nontransduced cells. Gene-marked Epstein-Barr
virus-specific CTL lines administered to immunocompromised patients
undergoing T-cell-depleted bone marrow transplantation were detectable
>18 months postinfusion (17). However, after 4 to 5 months the detectable frequencies of the transferred
neo-marked T cells were substantially lower than observed
early after the infusion, and the presence of the neo gene
could be demonstrated by PCR only after culturing samples of PBMC with
autologous Epstein-Barr virus-transformed B-LCL in vitro. Similarly, in
our study transferred HyTK-marked T cells declined to undetectable
levels more than 7 months postinfusion in the absence of a measurable
HyTK-specific CTL response. This decline may in part reflect dilution
due to repopulation of the lymphoid compartment following the
immunosuppressive therapy as well as a limited life span of the mature
gene-modified T cells. Thus, a decrease of the HyTK-marked T cells
below a threshold needed to maintain tolerance might explain the
failure to observe permanent tolerance with this approach.
A second explanation is suggested by recent studies which showed that
transferred allogeneic hematopoietic stem cells induced central
tolerance to donor antigen by negative selection (22). The
presence of systemic mixed donor/recipient hematopoietic microchimerism in solid organ recipients with stable long-term graft survival has led
to the hypothesis that the establishment of mixed microchimerism may
induce sustained tolerance (29, 43, 44, 48). However, it
has still not been determined whether the presence of donor hematopoietic cells is simply a marker of tolerance or is a
prerequisite for the development of tolerance.
In conclusion, we have shown that gene-modified T cells can induce
memory CD8+ CTL responses which persisted during and
following a potent immunosuppressive treatment consisting of low-dose
TBI (200 cGy), MMF, and CSP. However, this regimen prevented the
induction of a primary immune response against transgene-encoded
proteins and markedly prolonged the in vivo persistence of
gene-modified T cells. Sustained tolerance was not achieved, possibly
due to the requirement for the continued presence of the antigen. This
problem might be overcome by repeat administration of gene-modified T
cells at intervals to ensure that the antigen persists or by
introducing the gene into hematopoietic stem cells (18,
41).
| |
ACKNOWLEDGMENTS |
We thank Gary Millen, Robert G. Andrews, and the staff of the
University of Washington Regional Primate Research Center for assistance.
C.B. is supported by the Deutsche Krebshilfe, Dr.
Mildred-Scheel-Stiftung fuer Krebsforschung. H.-P.K. is a Markey
Molecular Medicine Investigator. This work was supported by National
Institutes of Health grants DK47754 (H.-P.K.), DK56465 (H.-P.K.),
AI43650 (S.R.R.), AI41754 (S.R.R.), CA18029 (S.R.R.), and NCVDG AI26503 (P.D.G.).
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Fred Hutchinson
Cancer Research Center, 1100 Fairview Ave. N., D1-100, Seattle, WA 98109-1024. Phone: (206) 667-4425. Fax: (206) 667-6124. E-mail: hkiem{at}fhcrc.org.
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Journal of Virology, January 2001, p. 799-808, Vol. 75, No. 2
0022-538X/01/$04.00+0 DOI: 10.1128/JVI.75.2.799-808.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
