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Blood, Vol. 95 No. 10 (May 15), 2000:
pp. 3071-3077
GENE THERAPY
Stable and functional lymphoid reconstitution of common cytokine
receptor  chain deficient mice by retroviral-mediated gene transfer
Claire Soudais,
Tsujino Shiho,
Lama I. Sharara,
Delphine Guy-Grand,
Tadatsugu Taniguchi,
Alain Fischer, and
James P. Di Santo
From the INSERM U429, Hôpital Necker, Paris, France;
Department of Immunology, University of Tokyo, Tokyo, Japan; and
Unité des Cytokines et Développement Lymphoide, Institut
Pasteur, Paris, France.
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Abstract |
Mutations in the gene encoding the common cytokine receptor gamma
chain ( c) are responsible for human X-linked severe
combined immunodeficiency disease (SCIDX1). We have used a
c-deficient mouse model to test the feasibility and
potential toxicity of c gene transfer as a therapy for
SCIDX1. A retrovirus harboring the murine c chain was
introduced into c-deficient bone marrow cells, which
were then transplanted into alymphoid RAG2/c
double-deficient recipient mice. Circulating lymphocytes appeared 4 weeks postgraft and achieved steady-state levels by 8 weeks. The mature
lymphocytes present in the grafted mice had integrated the
c transgene, expressed c transcripts, and
were able to proliferate in response to c-dependent cytokines. The c-transduced animals demonstrated (1)
normal levels of immunoglobulin subclasses, including immunoglobulin G1
(IgG1) and IgG2a (which are severely decreased in
c- mice); (2) the ability to mount an
antigen-specific, T-dependent antibody response showing effective in
vivo T-B cell cooperation, and (3) the presence of gut-associated
cryptopatches and intraepithelial lymphocytes. Importantly, peripheral
B and T cells were still present 47 weeks after a primary graft, and
animals receiving a secondary graft of c-transduced bone
marrow cells demonstrated peripheral lymphoid reconstitution. That
c gene transfer to hematopoietic precursor cells can
correct the immune system abnormalities in c- mice supports the feasibility of in vivo
retroviral gene transfer as a treatment for human SCIDX1.
(Blood. 2000;95:3071-3077)
© 2000 by The American Society of Hematology.
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Introduction |
One of the most studied cytokine receptor deficiencies
in man is X-linked severe combined immunodeficiency disease (SCIDX1), which results from defects in the common chain (c).
SCIDX1 accounts for 50%-60% of all cases of SCID (reviewed in 1) and is characterized by the complete absence of mature T and natural killer
(NK) cells, whereas B cells are frequently present in increased numbers. The thymus and peripheral lymphoid organs are severely hypoplastic in patients with SCIDX1, suggesting an early block in
T-cell differentiation. Following the co-localization of the gene
encoding the chain of the interleukin-2 receptor (IL-2R; now
denoted c) to the SCIDX1 locus at Xq12-13.1,
c mutations were identified in a number of these
patients,2 thereby demonstrating the essential roles of
c-dependent cytokines in human lymphoid development
(reviewed in 3).
The c chain was initially isolated as a functional
component of the intermediate and high-affinity IL-2R.4
Disruption of IL-2 mediated signaling, however, could not account for
the SCIDX1 phenotype, because IL-2 deficiency was compatible with T-cell development.5 Further studies established that
c also participated in the receptors for IL-4, IL-7,
IL-9, and IL-15 (reviewed in 6). The SCIDX1 phenotype, therefore,
results from combined defects in these 5 cytokine systems. A T-cell
developmental block similar to that seen in SCIDX1 can result from
IL-7R -deficiency in man,7 suggesting that IL-7 is
necessary for prothymocyte survival or expansion. In contrast, the
NK-cell differentiation block results principally from defects in IL-15
signaling pathways, because this cytokine is required to promote
NK-cell differentiation from bone marrow (BM) precursors.8
Concerning human B-cell development, it appears that lymphoid
precursors progress through c-dependent stages, but
c-independent pathways can compensate in SCIDX1.
Without treatment, patients with SCIDX1 suffer from severe, recurrent
infections, failure to thrive, and die within the first year of life.
The recent results of Buckley et al9 clearly demonstrate
that BM transplantation is the treatment of choice for SCIDX1, which is
curative for those patients who have an HLA-identical donor.
Haplo-identical transplants have also been performed, but they have a
lower success rate and are plagued by poor B-cell reconstitution,
thereby often necessitating long-term immunoglobulin replacement
therapy.9,10
Gene therapy remains an attractive alternative therapy for SCIDX1. In
principle, c-transduced hematopoietic precursors should demonstrate a marked selective advantage for lymphoid differentiation. This hypothesis has been supported by several in vitro studies, demonstrating that human c gene transfer into
Epstein-Barr virus-immortalized SCIDX1 B-cells lines could
reconstitute IL-2R signaling.11-13 Retroviral transduction
of SCIDX1 CD34+ BM precursor cells can permit NK- or T-cell
differentiation,14 although these results were obtained in
vitro under conditions that might not be attainable in vivo.
The existence of canine15 and murine16-18
models of c deficiency offer the possibility to test
gene therapy as an alternative treatment for SCIDX1.
c-Deficient mice are characterized by severe reductions
in B cells, NK cells, and gut-associated intraepithelial lymphocytes
(IEL). In contrast, mature activated T cells develop and accumulate in
c- mice, provoking inflammatory bowel
disease and splenomegaly.19,20 Despite this abnormal T-cell
development, c- mice are functionally
immunodeficient: (1) c- lymphocytes fail to
proliferate to mitogens or in mixed lymphocyte culture16,17; (2) c- mice fail
to reject tumors21; and (3) c-
mice fail to clear intracellular pathogens, such as Listeria monocytogenes and Toxoplasma gondii.22,23
c- Mice, therefore, recapitulate many
features of patients with SCIDX1. In this study, we have used
c- mice to examine the feasibility of in
vivo retroviral gene transfer to correct the immune system defects in
this SCIDX1 mouse model.
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Methods and materials |
Mice
Mice with a targeted c deletion16 were
maintained in specific pathogen-free conditions in an isolator barrier
facility (CNRS, Orleans, France) and were from the fourth generation
backcross to the C57Bl/6 background. A novel alymphoid (T-, B-, NK-)
mouse strain was generated by intercrossing recombinase activating
gene (RAG)-2 deficient mice, and
c-deficient mice (RAG2/c
mice21) were used as recipients for the transplants. All
mice were used at 4 to 12 weeks of age.
Cells
All cell lines were cultivated in DMEM supplemented with 10% fetal
calf serum (FCS) and penicillin/streptomycin. The derivation of the
retroviral packing cell line BOSC 23 and the transfection protocol used
to generate infectious retrovirus has been described in
detail.24 Transfectants expressing a soluble form of the murine stem cell factor and a hybridoma producing murine IL-6 were
kindly provided by Genetics Institute and Dr. Van Snick, respectively.
Cytokine-containing supernatants were used at optimal concentrations
after titration on appropriate cytokine-dependent cell lines.
Production of retroviral particles and infection of BM
precursors
Production of infectious, replication-defective ecotrophic
retrovirus using BOSC 23 cells was performed according to an
established protocol.24 A retroviral expression plasmid
(pMX25) using long terminal repeat (LTR) sequences from the
Moloney murine leukemia virus was engineered to express the murine
c chain. A full-length c complementary
DNA (cDNA) was amplified with the use of reverse transcription-polymerase chain reaction (RT-PCR) and
normal splenocyte RNA, subcloned into the EcoRI site of pMX, and fully
sequenced. Subconfluent BOSC 23 cells were transfected with 10 µg of
pMX-c by the calcium phosphate method.24
Retrovirus-containing supernatants were recovered 48 hours
posttransfection, filtered through 0.45 µm filters, and used directly
for infection of BM cells. Viral titer was determined using NIH3T3
cells as described.12
Bone marrow hematopoietic precursors were isolated following
intraperitoneal injection of 5-fluorouracil (5-FU at 150 mg/kg; 3 days
prior to harvest). Cells flushed from femora and tibias of 5-FU-treated
c- donor mice were cultured at
106 cells/mL in X-Vivo-10 medium (BioWhittaker)
supplemented with 5% FCS (Gibco BRL), murine stem cell factor (1/100
supernatant dilution), IL-6 (1/100 supernatant dilution), and Flt-3
ligand 100 ng/mL (kindly provided by Immunex Corp) with an equal volume of retroviral supernatant in the presence of 10 µg/mL of Polybrene (Sigma) on fibronectin-coated tissues culture plates at 37°C in 5%
CO2 fully humidified incubators. Purified whole fibronectin (Sigma; dissolved at 100 µg/mL in phosphate-buffered saline) was used
to coat plates for 2 hours at room temperature. This infection protocol
was repeated daily for 2 more days; supernatants were removed, and
nonadherent cells recovered and replated in fresh virus-containing
medium. A mock transduction protocol was performed in exactly the same
fashion, except that BOSC 23 cells received the parental pMX vector.
Recovered nonadherent BM cells (2 × 106) were grafted
into the tail vein of irradiated (0.3 Gy) RAG2/c double
mutant recipient animals. Total BM cells from primary recipient mice
were used for secondary transfers.
Cell isolation, in vitro proliferation analysis, and
immunofluorescence
Lymphoid organs were removed, and single-cell suspensions were
prepared using a mesh filter. Hepatic lymphocytes are a rich source of
NK cells and were analyzed as described.21 Splenocytes were
cultured at 2 × 105 cells/well in flat-bottom 96 well
plates in DMEM supplemented with 10% FCS with or without concanavalin
A (Con A; 2.5 µg/mL) and c-dependent cytokines (IL-2
or IL-7 at 20 ng/mL) for 72 hours. Thymocytes were similarly cultured
in U-bottom 96 well plates with or without phorbol myristate acetate
(PMA) (10 ng/mL) and IL-4 (100 ng/mL). Cells were pulsed
with 0.5 µCi of 3H-thymidine during the final 16 hours of culture.
Immunofluorescence analysis was performed as
described.16,21 Antibodies against the following cell
surface antigens were used for immunofluorescence analysis (all
from Pharmingen) as FITC-, PE-, or TRICOLOR conjugates: CD4, CD8,
TCR , TCR , B220, immunoglobulin M (IgM), IgD, and
DX5. A combination of anti-TCR and anti-B220 antibodies were
used to sort splenocytes, using a FACStar+ cytometer prior
to DNA isolation.
PCR detection of the c gene, transgene, and
transcripts
For detection of the retrovirally transduced c
transgene, total splenocyte or sorted lymphocyte DNA was extracted,
using proteinase K in polymerase chain reaction (PCR) buffer containing 0.1% Tween 20. Following enzyme inactivation, a transgene specific PCR
was performed, using exon 6 (5'-CTTCCTTGTTTGCACTGG-3') and exon 8 (5'-GGGGAGGTTAGCGTCACTTAGGAC-3') primers amplify a 400-base pair (bp) c cDNA fragment. For RT-PCR, total peripheral
blood lymphocyte RNA was converted into first strand cDNA using
standard procedures, and amplification was performed using the primers described above.
T-dependent antigen-specific immunoglobulin responses and total
immunoglobulin levels
Animals were immunized intraperitoneally with 100 µg of
alum-precipitated nitrophenyl (NP)-conjugated bovine serum albumin (NP-BSA) and 109 inactivated Bordatella pertussis.
Pre-immune, day 7, and day 14 sera were collected. Levels of
NP-specific IgM or IgG were determined by enzyme-linked immunosorbent
assay (ELISA) as described.26 Briefly, ELISA plates were
coated with 5 µg/mL NP-BSA overnight at 4°C and blocked with 5%
BSA. Sera and standards were serially diluted and incubated for 1 hour
at 37°C. Specific bound antibodies were revealed using
isotype-specific anti-mouse antibodies directly conjugated to alkaline
phosphatase followed by conversion of the chromogenic substrate p-NPP
(Sigma). Total serum immunoglobulin isotype levels were determined
using an ELISA kit (Pharmingen) according to the manufacturer's
instructions. Statistical analysis was performed using the Student
t test.
Histological analysis
Tissues (1-cm piece of small bowel) were fixed in Carnoy solution
and embedded in paraffin. Sections were stained with methyl/pyronin or
by the periodic acid-Schiff reaction. Intraepithelial lymphocytes were
enumerated as described.16
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Results |
Study design and peripheral lymphoid reconstitution in
c-transduced mice
Bone marrow cells were harvested from 5-FU-treated
c- mice and infected with
retroviral particles (titer: 106/mL) harboring the murine
c chain driven by the MoMLV LTRs.25 Three
1-day infection cycles were performed before transfer into alymphoid
recipient animals (a new strain deficient in both the RAG2 and
c genes: RAG2/c mutant mice).
RAG2/c rather than c- mice
were used as hosts because the latter demonstrate abnormal T-cell
development that has been shown to perturb steady-state hematopoiesis
and provoke splenomegaly.19 RAG2/c mice have no mature T, B, or NK cells but have otherwise normal hematopoietic parameters,21 thereby permitting donor lymphopoeisis to be
determined in an unambiguous fashion. RAG2/c mice
(n = 7) that had been transplanted with retrovirally
infected c- BM cells will be
referred to as "c-transduced" mice. Lymphoid reconstitution in an equivalent number of RAG2/c mice
that had been transplanted with mock infected
c- BM cells gave an immunophenotype and
function identical to nonmanipulated c- mice
(data not shown). We have previously demonstrated lymphoid reconstitution in the RAG2/c strain, using normal
BM-derived hematopoietic precursors.21
Peripheral lymphoid cells were analyzed in c-transduced
mice as well as c, RAG2/c, and control
C57Bl/6 (c+) mice. As previously
reported,16-18 c- mice developed
some mature T cells but very few B cells (Figure 1A), whereas circulating lymphoid cells
were not detected in the RAG2/c mutants. In
c-transduced mice, mature T and B cells could be
detected as soon as 4 weeks after the transplant, and, by 12 weeks
postgraft, steady-state levels had been reached (Figure 1A). The
kinetics and magnitude of lymphoid reconstitution observed in
c-transduced mice were comparable to that observed
following transplantation of unmanipulated or mock-infected normal BM
into RAG2/c mice21 (Table
1 and data not shown). Among individual c-transduced mice, some variation between relative
proportions of T and B cells could be documented, yet all animals
showed peripheral lymphoid reconstitution (Table 1). These results
contrast sharply with the immune phenotype in
c-deficient mice, whereby B lymphocyte numbers decline
with age.19
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| Fig 1.
Lymphoid reconstitution in c-transduced
mice.
(A) Flow cytometric analysis of peripheral blood T and B cells, using
FITC-conjugated anti-TCR and PE-conjugated anti-B220 antibodies.
In these experiments, lymphocytes expressing T- or B-cell markers are
calculated as a percentage of total nucleated cells to emphasize the
kinetics of lymphoid reconstitution. (B-E) Analysis of c
transgene integration and expression in c-transduced
animals. Polymerase chain reaction (PCR) was performed using exon 6- and exon 8-specific primers (B). The endogenous c locus
(0.8 kilobase [kb]) is amplified in wild-type mice but
not in c- mice in which exon 6 has been
deleted.16 c-Transduced mice show a 0.4-kb
product derived from the integrated c transgene at 23 weeks postgraft. (C) Expression of the c transgene was
detected by RT-PCR from peripheral blood cells of control
and c-transduced animals at 7 weeks postgraft.
Contaminating genomic DNA in the c+ sample gives rise to
a PCR product in the absence of reverse transcription. (D) Schematic of
the retroviral construct used with the location of transmembrane (exon
6) and intracytoplasmic (exon 8) primers. (E) Expression of
c on c+,
c-, and c-transduced cells.
Staining of total thymocytes with isotype control monoclonal antibody
(dotted line) and c-specific monoclonal antibody (solid
line) are shown.
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Integration and expression of retrovirally transduced
c transgene
To verify the presence and expression of the retroviral
c transgene in c-transduced animals,
genomic DNA and RNA were prepared from either total peripheral blood
cells, total splenocytes, or from sorted splenic T and B cells. A PCR
was then performed, using primers specific for exons 6 and 8 of the
c gene (Figure 1B-D). These primer pairs amplify a
0.8-kilobase (kb) fragment from the wild-type c locus
and a 0.4-kb fragment from the c cDNA transgene or
endogenous transcript, but they do not amplify the targeted c locus lacking exon 6.16
A PCR product (0.4 kb) corresponding to the size expected for
c cDNA transgene was detected, using DNA derived from
peripheral blood of all c-transduced mice (data not
shown). Genomic retroviral integration could be demonstrated in total
splenocyte DNA from 7 of 7 c-transduced animals at 7 to
47 weeks postgraft (Figure 1B; data not shown) and was also detected in
sorted splenic lymphocytes from 2 of 2 animals tested at 23 weeks
postgraft. No amplification products were found in mock-transduced
animals, and, in C57Bl/6 mice, only the expected fragment corresponding
to the wild-type c locus was detected (Figure 1B; data
not shown). Moreover, c-transduced animals (5 of 5 tested) expressed transgene-specific c transcripts of
the expected size in peripheral blood cells (Figure 1C). Finally, c expression could be detected on the surface of
c-transduced cells (Figure 1E). We conclude that
retroviral infection of c- BM precursors can
generate peripheral lymphoid cells with stable integration and
expression of the transduced c transgene.
Phenotype and function of peripheral lymphocytes from
c-transduced mice
Splenic-cell populations were further characterized in
c-transduced animals. The total number of splenocytes
was significantly increased in c-transduced animals
compared with RAG2/c recipients or to the
c- donor mice (Table 1). A normal ratio of
CD4- to CD8-expressing cells was observed in 7 of 7 c-transduced animals (Figure
2A); these cells expressed normal levels of
TCR (data not shown). Splenocytes from these mice demonstrated a
normal pattern of IgM and IgD expression, indicating the presence of
both newly generated and recirculating mature B cells (Figure 2B; these
cells co-expressed CD19, data not shown). In contrast, CD3+
TCR T cells were detected in 3 of 7 c-transduced
animals (data not shown), while DX5+ TCR-
NK cells were present in 3 of 7 c-transduced mice
(Figure 2C). One c-transduced mouse harbored both
TCR cells and NK cells (data not shown). Why T cells and
NK cells were found in only a fraction of
c-transduced mice remains unclear but may be related to
the level or timing of transgene expression by the retroviral promoter.
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| Fig 2.
Characterization of splenic and gut lymphocytes from
c-transduced mice.
Flow cytometric analysis of splenocytes using (A) FITC-conjugated
anti-CD8 and PE-conjugated anti-CD4 antibodies or (B) FITC-conjugated
anti-immunoglobulin D (IgD) and PE-conjugated anti-IgM antibodies. (C)
Analysis of hepatic lymphocytes using FITC-conjugated anti-DX5 (natural
killer cell specific) and PE-conjugated anti-TCR antibodies.
Positive cells are expressed as percentage of gated lymphocytes. (D)
Representative sections of small bowel epithelium in a
c-transduced mouse (Periodic Acid Shiff staining,
× 250). Intraepithelial lymphocytes (arrows), normal cellularity of
the lamina propria, and lymphoid cryptopatches (large arrow) were
identified that were indistinguishable from control
c+ animals16 (and data not
shown).
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To determine whether peripheral T lymphocytes present in
c-transduced animals expressed functional
c-containing receptor complexes, splenocytes were
stimulated with Con A alone or in the presence of the
c-dependent cytokines IL-2 or IL-7 (Table 2). We have previously demonstrated that
IL-2 and IL-7 do not promote the proliferation of
c-deficient cells.16 In contrast, IL-2 or
IL-7 stimulated the Con A mitogenic response of splenocytes from 3 of 3 c-transduced animals (Table 2). Thymocytes from c-transduced mice also responded to the combination of
IL-4 plus PMA, unlike c- thymocytes (Table
2). These results demonstrate that c gene transfer can
restore expression of functional IL-2, IL-4, and IL-7 receptors on T
cells from c-transduced animals.
B-cell responses in c-transduced animals
To assess B-cell function in c-transduced mice, we
first analyzed steady-state circulating plasma immunoglobulin levels in animals >8 weeks posttransplant. c- mice
exhibit abnormal serum immunoglobulin levels, reflecting defective
B-cell differentiation.16,17 In particular,
c- mice have extremely reduced levels of
IgG1 and a 2 log reduction in the concentrations of serum IgG2a (Figure
3A). Following c gene
transfer, we found a normal concentration of all serum immunoglobulin levels tested in c-transduced animals (Figure 3A).
Levels of IgG1 and IgG2a were not significantly different from control
C57Bl/6 mice (P = .21 for IgG1 and P = .35 for
IgG2a). Sera from RAG2/c mice were negative for all
immunoglobulin subclasses.
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| Fig 3.
B-cell responses in c-transduced animals.
(A) Serum immunoglobulin isotype concentrations were established by
enzyme-linked immunosorbent assay (ELISA) using purified immunoglobulin
standards. Each dot represents 1 mouse (7 animals for each strain
listed, except for RAG2/c mice). (B) Normal T-dependent
antigen immunoglobulin responses in c-transduced
animals. Following immunization with nitrophenyl-conjugated bovine
serum albumin, circulating NP14-specific immunoglobulin M (IgM) and IgG
antibodies were determined by ELISA for control (black square), 3 independent c-transferred animals (open symbols), and
c- mice (-X-). Only 1 each of control and
c- mice is shown for simplicity; 3 additional mice of each genotype gave similar results.
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B-cell immunoglobulin responses were evaluated, following immunization
with the T cell-dependent antigen NP-BSA. As shown in Figure 3B, normal
titers of circulating anti-NP-BSA-specific IgM antibodies could be
detected in c-transduced animals by day 7 postimmunization and persisted at day 14. Moreover, switched IgG
antibodies could be detected in these mice at day 14. In contrast, immunization of c- mice, using either
T-dependent or T-independent antigens, fails to elicit any
antigen-specific immunoglobulin (Figure 3B; Vosshenrich et al,
submitted). Taken together, these data demonstrate that c-transduced animals harbor mature B cells and T cells
capable of functional immune responses.
Restoration of the gut-associated lymphoid cells following
c gene transfer
Distinct morphological structures located in between the intestinal
crypts (cryptopatches) contain cells with an immature lymphoid
phenotype (c-kit+, IL-7R+) that appear to
play a role in the generation of some IEL T-cell subsets.27,28 Cryptopatch generation requires the IL-7R
chain28 or the c chain (our unpublished
observations). The development of all IEL subsets is c
dependent as well.16
Gut-associated lymphoid cells developed in c-transduced
mice. Cryptopatches were detected in 7 of 7 c-transduced
animals (Figure 2D). Their morphological structure was comparable to
that of control C57Bl/6 mice (data not shown). In addition,
IELs could be detected in the c-transduced animals
(Figure 2D, Table 1), whereas IELs have never been observed in
c- mice.29 Together, these
observations suggest that c gene transfer into
hematopoietic precursors capable of generating intestinal lymphoid
cells had occurred.
Stability of retroviral gene expression in
c-transduced animals
Two c-transduced animals were analyzed at 40 or 47 weeks after the initial transplant. In these animals, we were able to detect normal percentages of circulating mature B and T
cells (Figure 4). The
retrovirally transduced c transgene was detected in
splenocyte DNA from these mice, and development of gut-associated lymphoid cells had occurred (data not shown). No pathological effects
of gene transfer were observed in c-transduced animals analyzed during the course of this study.
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| Fig 4.
Stability of lymphoid reconstitution in
c-transduced mice.
Flow cytometric analysis of peripheral blood T and B cells using
FITC-conjugated anti-TCR and PE-conjugated anti-B220 antibodies.
Positive cells are expressed as percentage of gated lymphocytes. The
left panel demonstrates long-term peripheral reconstitution 47 weeks
posttransfer with normal percentage of T and B cells. The center panel
shows a c-transduced animal at 8 weeks postgraft. Bone
marrow cells from this mouse were transferred into a secondary
irradiated recipient, which, after 8 weeks, demonstrated normal
peripheral reconstitution (right panel).
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To determine whether stable integration of the c
transgene could be achieved in c-
hematopoietic stem cells, we transplanted 106 BM cells from
3 different c-transduced mice (at 8 to 12 weeks postgraft) into secondary RAG2/c recipient mice. The
primary c-transduced mice demonstrated normal
circulating B and T cells at this time, and, on secondary transfer, the
c-transduced BM inoculum could again give rise to normal
percentages of mature B and T cells (a representative example is shown
in Figure 4). A total of 8 mice were analyzed up to 23 weeks following
secondary transfers, and the kinetics and stability of peripheral
lymphoid reconstitution were similar between primary and secondary
recipients (data not shown).
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Discussion |
In this study we have shown that ex vivo c gene
transfer into hematopoietic precursor cells from
c-deficient mice can lead to a correction of the immune
deficiency without obvious side effects. Following transfer of the
retrovirally transduced c- BM cells to
alymphoid recipients, we found that engrafted animals generated
peripheral lymphoid cells that stably integrated and expressed the
c transgene. Both mature B cells and T cells were present in the recipients: c-transduced lymphocytes
responded to c-dependent cytokines and were capable of
generating a cooperative (B-T cell) immune response following antigen
immunization. Finally, c-transduced mice showed
development of gut-associated lymphoid cells, the presence of which is
entirely dependent on expression of the c
chain.16 Taken together, these results demonstrate the
feasibility of c gene transfer ex vivo and fulfill an
important prerequisite for further clinical studies of c
gene therapy for patients with SCIDX1.
The retroviral infection protocol used in our model was not aimed at
optimizing gene transfer conditions. Nevertheless, cytokine stimulation
of BM progenitor cells followed by 3 cycles of infection (using viral
supernatants and fibronectin-coated plates) was found to work
efficiently as previously documented.30-32 Importantly, this protocol closely matches current conditions established for treatment of patients with SCIDX1 by BM transplantation or ex vivo gene
transfer, including noncytoablative host conditioning.9 The
observations that distinct T-cell subsets, B cells, and IEL-cell populations were stably reconstituted in c-transduced
mice up to 47 weeks postprimary transplant as well as on secondary
marrow transplantation (up to 23 weeks after transfer) strongly suggest that early hematopoietic progenitor cells with self-renewal capacity were successfully infected. It should be noted that both primary and
secondary RAG2/c recipients were not subjected to lethal irradiation, so that competition between endogenous hematopoietic cells
and transduced donor cells could well have occurred.
A human autosomal SCID syndrome due to JAK-3 deficiency has been
described that is immunologically identical to SCIDX1,33,34 thereby highlighting the requirement for JAK-3 activation following c-receptor signaling. Recently, a mouse model of JAK-3
deficiency was successfully treated by ex vivo gene transfer with the
generation of peripheral T and B cells that were functionally
responsive to c-dependent cytokines.35 Thus,
ex vivo retroviral gene transfer appears capable of correcting immune
deficiencies secondary to c or JAK-3 defects.
It is important to note that, in these experiments, the transduced
genes were constitutively expressed under the control of the viral LTR.
Dysregulated c or JAK-3 signaling could in theory lead
to autonomous cell activation caused by receptor or kinase overexpression. However, in the case of c-transduced
mice, no evidence of myelo- or lymphoproliferation or autoimmune
manifestations was observed in the long-term reconstituted animals. The
absence of side effects could reflect the low level of
c expression detected in the treated animals. Still, the
lymphoid system of c-transduced mice appeared functional
by a number of criteria (in vitro proliferative responses, specific
immunoglobulin production following immunization), suggesting that
high-level c expression may not be a requisite for
immune reconstitution. Because c is constitutively
expressed in multiple hematopoeitic lineages (reviewed in 36), a
potential protein excess might not have negative consequences. These
results suggest that (1) regulated transgene expression may not be
required for the generation of functional lymphoid cells from
c-hematopoietic precursors and that (2)
retroviral gene transfer to correct deficiencies in the
c-signaling pathways may have low-potential toxicities,
which is an important consideration for any new therapeutic approaches.
Although secondary extinction of retroviral transgenes has been
reported,37-39 extinction of the c transgene
was not observed in this study, including mice receiving secondary
transplants and analyzed 23 weeks later. Whether extinction is a
randomly occurring event or requires a particular chromatin
conformation around the proviral integration site is not known. It is,
therefore, difficult to predict from this animal model of SCIDX1 what
the outcome of gene therapy in patients with SCIDX1 will be with regard to long-term expression from retroviral vectors. Natural selection of
c-expressing cells may be an important factor in
preserving transgene expression in vivo following gene transfer. It is
worthwhile noting that c-transduced SCIDX1 Epstein-Barr
virus-transformed B-cell lines were maintained for more than 1 year in
culture without evidence of loss of the c transgene
expression.12
Spontaneous in vivo reversion of inherited mutations has been observed
in patients with ADA and SCIDX1 (reviewed in 1). In the
latter case, the c reversion resulted in a significant
and long-lasting correction of the T-cell deficiency. This result strongly suggests that a c+ lymphoid
precursor carries a major survival and/or growth advantage over
c- cells in vivo and are in agreement with
the original observations of skewed X-inactivation patterns in SCIDX1
obligate female carriers made by Puck et al.40 These
observations provide a logical basis to hypothesize that a selective
advantage would be conferred to corrected progenitor cells in patients
with SCIDX1. The results obtained in c- and
JAK3-deficient mice support the notion that human SCID syndromes represent a rather favorable model for treatment by ex vivo gene transfer.
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Acknowledgments |
We are grateful to Dr. Francois Huetz for providing immunization
reagents, Fabian Gross for fibronectin, Françoise Selz for cell
sorting, and Michele Malassis for technical help.
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Footnotes |
Submitted July 20, 1999; accepted December 21, 1999.
Supported by grants from the Institut National de la Santè et de
la Recherche Medicale (INSERM), Ligue Contre le Cancer, and Association
Francaise contre les Myopathies.
Reprints: James P. Di Santo, Unité des Cytokines et
Développement Lymphoide, Institut Pasteur, 25 rue du Dr Roux, F-75 724 Paris, France; e-mail: disanto{at}pasteur.fr.
The publication costs of this
article were defrayed in part by
page charge payment. Therefore,
and solely to indicate this fact,
this article is hereby marked
"advertisement"
in accordance with 18 U.S.C.
section 1734.
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Abstract
Introduction