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Blood, Vol. 94 No. 9 (November 1), 1999:
pp. 3027-3036
Restoration of Lymphoid Populations in a Murine Model of X-Linked
Severe Combined Immunodeficiency by a Gene-Therapy Approach
By
Mindy Lo,
Michael L. Bloom,
Kazunori Imada,
Maria Berg,
Julie M. Bollenbacher,
Eda T. Bloom,
Brian L. Kelsall, and
Warren J. Leonard
From the Laboratory of Molecular Immunology, National Heart, Lung,
and Blood Institute, National Institutes of Health (NIH), Bethesda;
Hematology Branch, National Heart, Lung, and Blood Institute, NIH,
Bethesda; the Center for Biologics Evaluation and Research, Food and
Drug Administration, Bethesda; and the Laboratory of Clinical
Investigation, National Institute of Allergy and Infectious Diseases,
Bethesda, MD.
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ABSTRACT |
X-linked severe combined immunodeficiency (XSCID) is a
life-threatening syndrome in which both cellular and humoral immunity are profoundly compromised. This disease results from mutations in the
IL2RG gene, which encodes the common cytokine receptor  chain, c. Previously, we generated
c-deficient mice as a murine model of XSCID. We have now
used lethally irradiated c-deficient mice to evaluate a
gene therapeutic approach for treatment of this disease. Transfer of
the human c gene to repopulating hematopoietic stem
cells using an ecotropic retrovirus resulted in an increase in T cells,
B cells, natural killer (NK) cells, and intestinal intraepithelial
lymphocytes, as well as normalization of the CD4:CD8 T-cell ratio and
of serum Ig levels. In addition, the restored cells could proliferate
in response to interleukin-2 (IL-2). Thus, our results provide added
support that gene therapy is a feasible therapeutic strategy for XSCID.
Moreover, because we used a vector directing expression of human
c to correct a defect in c-deficient mice, these data also indicate that human c can
cooperate with the distinctive cytokine receptor chains such as
IL-2R and IL-7R to mediate responses to murine cytokines in vivo.
This is a US government work. There are no restrictions on its use.
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INTRODUCTION |
X-LINKED SEVERE COMBINED immunodeficiency
(XSCID) is a life-threatening disease characterized by
greatly diminished numbers of T and natural killer (NK)
cells.1-3 Although B cells are present in normal numbers in
XSCID patients, they are nonfunctional.1-3 XSCID is caused
by mutations in the common cytokine receptor chain,
c,4 which is a component of the receptors
for interleukin-2 (IL-2), IL-4, IL-7, IL-9, and IL-15.5-11
The simultaneous inactivation of these 5 different cytokine systems
therefore helps to explain the severity of the immunological defects in
this disease, with defective IL-7 signaling likely explaining the
defective T-cell development,12 and defective IL-15
signaling explaining the defective NK-cell development.12a
XSCID is the most common form of SCID, accounting for approximately
half of all cases of SCID.1-3 Although most children with
XSCID can be successfully treated by haploidentical bone marrow
transplantation, in many of these individuals, the donor T cells
engraft, but B cells do not, resulting in clinically relevant
hypogammaglobulinemia that necessitates chronic intravenous
gammaglobulin therapy.13,14 Therefore, the development of
gene therapy for individuals with this disease could represent a
substantial therapeutic advance.
Previously, our group and others have generated
c-expressing retroviral vectors, which can infect
mammalian cells in vitro.15-18 More recently, investigators
have used retroviral transduction of bone marrow to direct
c expression in peripheral lymphocytes in normal
dogs.19 Finally, c has been transduced into
bone marrow from XSCID hematopoietic stem cells with the maturation of
CD34+ bone marrow cells into double positive
(CD4+CD8+) and single positive
(CD4+CD8 and
CD4CD8+) lymphocytes in a hybrid
human/mouse fetal thymic organ culture.20 As a logical next
step towards the eventual development of gene therapy for this disease,
we attempted to correct the immunological defect in mice in which
c had been deleted by homologous recombination. Such an
approach has been successfully performed for mice lacking expression of
Jak3.21 Jak3 deficiency is an autosomal recessive form of
SCID that is clinically and immunologically similar to XSCID.22,23
Like humans with XSCID, c-deficient mice exhibit marked
T-cell and NK-cell defects.24-26 Examination of
c-deficient mice has also shown the absence of
"natural" NK1.1+ T cells as well as  T cells,
including, for example, dendritic epidermal T cells. In addition, these
mice lack both  and intestinal intraepithelial lymphocytes
(IELs).24,25 A major difference between humans and mice
lacking c expression is that B cells develop in humans
with XSCID (although they are dysfunctional), but B-cell development is
markedly diminished in the c-deficient mice.24-26 This latter difference appears to result from
the importance of IL-7 signaling for murine B-cell development, whereas
this function in humans is either served by another cytokine or is redundant.12 Thus, while the murine syndrome is somewhat
different from human XSCID, the c-deficient mice
nevertheless are valuable models for determining whether a gene
therapeutic approach can result in correction of the defects in
lymphoid development and T-cell function. We chose to use an ecotropic
retrovirus directing expression of human c to attempt
gene therapy in the c-deficient mice. Transduction of
donor c-deficient bone marrow with this virus followed
by bone marrow transplantation into c-deficient mice
increased expression of T cells, B cells, and NK cells. This establishes the potential efficacy of gene therapy and also
demonstrates the ability of human c to cooperate with
the relevant murine cytokines and their distinctive receptor components.
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MATERIALS AND METHODS |
Retroviral producer cell lines.
The full-length human c cDNA was excised from pCRScript
using BsaI and BamHI and cloned into the NcoI
and BamHI sites of the pMMP vector to generate
pMMP-c; the construction of pMMP will be reported in
detail elsewhere by Drs Jeng-Shin Lee and Richard C. Mulligan (Harvard
Medical School, Boston, MA), who generously provided this
vector to us. Briefly, the vector uses the myeloproliferative sarcoma
virus (MPSV) long terminal repeats (LTRs) and glutamine
tRNA primer binding site (PBSQ) instead of the wild-type proline PBS.
Both modifications were introduced to improve gene
expression.27 pMMP-c was transfected
together with pCDNA3.1 (which carries the Neomycin resistance gene;
Invitrogen, Carlsbad, CA) using the calcium phosphate
method into the Phoenix ecotropic packaging cell line (provided by Dr
Garry Nolan, Stanford University, Stanford, CA; see
http://cmgm.stanford.edu/micro/fac/nolan.html) and the 293 SPA
amphotropic packaging cell line (provided by Dr H.L. Malech, National
Institutes of Health [NIH]), and stable clones were selected in G418
(Life Technologies, Grand Island, NY). Supernatants from
isolated, stable producer Phoenix and 293 SPA clones were screened for
their ability to induce expression of human c in NIH3T3
cells, as assessed by flow cytometric analysis.
Bone marrow transduction and transplantation.
The c-deficient mice in this study have been previously
described25,28,29 and were back-crossed for more than 10 generations to C57BL/6 mice. Wild-type C57BL/6 or
c-deficient donor mice (6 to 10 weeks old) were injected
intravenously with 5-fluorouracil (150 mg/kg body weight) 48 hours
before harvesting of bone marrow. Marrow was flushed from both lower
limbs and prestimulated for 48 hours in bone marrow medium (Dulbecco's
modified Eagle's medium [DMEM] containing 15% fetal bovine serum, 4 mmol/L L-glutamine plus 20 ng/mL murine IL-3, 50 ng/mL murine IL-6, and
100 ng/mL murine stem cell factor (cytokines were all from
PeproTech, Rocky Hill, NJ). Bone marrow cells were then
cocultured for 48 hours with the Phoenix producer cells in the same
medium with 6 µg/mL polybrene (Sigma, St Louis, MO) at
32°C. Cells were then transferred to plates coated with RetroNectin
(recombinant human fibronectin fragment CH-296, 20 µg/cm2; TaKaRa Biomedicals, Shiga, Japan) and infected
for 24 hours at 32°C with viral supernatant. The viral supernatant
was generated by culturing a human c virus-producing
Phoenix cell clone in bone marrow medium for 16 hours. After a further
24-hour recovery period in fresh bone marrow medium, cells were
collected and approximately 1 × 106 cells were
injected intravenously into 6- to 8-week-old recipient C57BL/6
c-deficient mice that had been lethally irradiated with 800 rads. Mock-transduced mice were handled identically except that
bone marrow cells were cocultured with untransfected Phoenix cells that
were not producing virus. Bone marrow cells from wild-type mice were
just maintained in culture and were not exposed to Phoenix cells or virus.
Transduction of human Epstein-Barr virus
(EBV)-transformed B-cell line from a patient with XSCID.
EBV-transformed B cells from a patient with XSCID (provided by Dr J.M.
Puck, NIH, IL-2RG database cDNA number 830del 4[3] containing a
deletion/frameshift at Leu 272; see
http://www.nhgri.nih.gov/DIR/LGT/SCID/scid_query_result.hts?ExonIntronNumber=6) were maintained in RPMI 1640 medium supplemented with 2 mmol/L L-glutamine, 10% fetal bovine serum, 100 U/mL penicillin, and 100 µg/mL streptomycin. The cells were transduced with the
c-expressing amphotropic retrovirus, which was collected
after 12 to 16 hours culture of the confluent 293 SPA virus-producing
clone. The infection was performed during 3 24-hour cycles of
incubation on RetroNectin-coated tissue culture dishes at 32°C in
the presence of 6 µg/mL of polybrene. The cells were allowed to
recover in growth medium for 30 hours before fluorescence-activated
cell sorting (FACS) analysis or stimulation with IL-2 or IL-4.
Polymerase chain reaction (PCR) detection of transduced gene in
peripheral blood.
A total of 50 µL of peripheral blood was obtained by retroorbital
bleed, and DNA was isolated using the Instagene Whole Blood kit
(Bio-Rad, Hercules, CA). Primers used to amplify the human c cDNA were as follows: forward primer (M10),
5'-CTCTTATTCCTGCAGCTGCC-3'; reverse primer (M19),
5'-CGTTCCAGCCAGAAATACAC-3'. Amplification of human
c was performed using 10 µL DNA per reaction for 40 cycles of 45 seconds at 94°C, 45 seconds at 58°C, and 90 seconds at 72°C.
Flow cytometric analysis.
Fresh cells from thymus, spleen, and bone marrow were stained and
analyzed on a FACSort (Becton Dickinson, San Jose, CA) using CellQuest
software. Splenocyte cell suspensions were treated with ACK lysing buffer to remove red blood
cells. Some splenocytes were also removed for 48-hour stimulation in
6-well plates coated with 10 µg/mL of anti-mouse CD3 (145/2C11
monoclonal antibody [MoAb]). Cells were then stained with the
following antibodies (PharMingen, San Diego, CA): fluoroscein
isothiocyanate (FITC)-conjugated anti-mouse IgM, Cy-chrome-conjugated
anti-mouse CD45R/B220, FITC-conjugated anti-mouse CD25 (anti-IL-2R,
PC61), phycoerythrin (PE)-conjugated anti-mouse CD62L (L-selectin),
Cy-chrome-conjugated anti-mouse CD4 (L3T4), allophycocyanin
(APC)-conjugated anti-mouse CD8 (Ly-2) (53-6.7), APC-conjugated
anti-mouse CD3 (145-2C11), PE-conjugated anti-mouse NK-1.1,
Cy-chrome-conjugated anti-mouse T-cell receptor (TCR)
(H57-597), and PE-conjugated TUGh4 MoAb (against human c). To eliminate possible binding to Fc receptors,
anti-mouse CD16/CD32 MoAb (2.4G2) was added during staining.
EBV-transformed B cells that were not transduced or transduced with the
c retrovirus were stained with PE-conjugated mouse
anti-human c MoAb AG184 or a PE-conjugated mouse
isotype-matched control MoAb (PharMingen).
Quantitation of serum Igs.
Serum was obtained from mice at the time of sacrifice by retroorbital
bleed. Total serum IgA, IgG, and IgM levels were measured by sandwich
enzyme-linked immunosorbent assay (ELISA) using Ig quantitation kits
according to the manufacturer's instructions (Bethyl Laboratories,
Montgomery, TX). Briefly, 96-well, flat-bottom ELISA plates (Immunon
4TM; Dynex, Chantilly, VA) were coated with a polyclonal goat
anti-mouse Ig Fc-specific antibody. Serum samples or a standard
reference serum were serially diluted in phosphate-buffered saline
(PBS) with 1% (wt/vol) bovine serum albumin (Sigma) and added to the
plates. Bound Igs were detected using horseradish peroxidase
(HRP)-labeled goat anti-mouse Ig Fc-specific antibodies followed by
addition of the HRP-substrate o-phenylenediamine (1 mg/mL; Sigma) and
H2O2 (0.03%) in phosphate-citrate buffer (pH 5.0). Serum Ig levels were determined by comparing the absorbance values (450 nm) of the samples with the values from the linear portion
of the curve generated with the reference serum.
Splenocyte proliferation.
Splenocytes prepared as above were plated in triplicate in a 96-well
plate at 2 × 105 cells/well in RPMI 1640 medium
containing 10% fetal bovine serum, 2 mmol/L L-glutamine, and
antibiotics. The cells were stimulated for 48 hours at 37°C with
and without human IL-2 (2 nmol/L, kindly provided by Hoffmann
LaRoche, Nutley, NJ). Cells were then pulsed with 1 µCi/well of methyl-3H-thymidine (NEN, Boston, MA) for 8 hours. 3H-thymidine uptake was then determined using a beta
plate counter.
Transfection of 293T cells.
293T cells (a human embryonic kidney cell line expressing SV40 large T
antigen) were transfected by the calcium phosphate method (transfection
kit from 5'-3', Inc, Boulder, CO). One million cells were
plated into 100-mm tissue culture dishes 24 hours before transfection
with 2 µg of murine IL-7R, 2 µg of Jak3, 0.5 µg each of Stat5a
and Stat5b, and 2 µg of murine or human c expression vectors. Each of these cDNAs was cloned in pME18S, a eukaryotic expression vector in which transcription is driven by the SR promoter.30 Twenty-four hours after transfection, cells
from each dish were split between 2 × 100 mm dishes and 24 hours
later, cells were either stimulated or not stimulated with murine or human IL-7 for 10 minutes at 37°C, washed with cold PBS, and
nuclear extracts were prepared.
Isolation of murine IL-7R cDNA.
The murine IL-7R cDNA was generated by PCR-mediated amplification of
mRNA from C57BL/6 lymphocytes and from 70Z/3 cells using primers based
on the published murine IL-7R sequence.31 DNA sequencing
of our cDNAs in each case showed 5 nucleotide alterations corresponding
to 4 amino acid changes, as compared with the previously reported
murine IL-7R sequence.31 Our sequence has been submitted to GenBank (Accession no. AF078906).
Electrophoretic mobility shift assays (EMSAs).
Whole-cell or nuclear extracts were prepared as previously
described.32 The -casein, PRRIII, and FcRI probes
were labeled with 32P-deoxycytidine triphosphate
(dCTP) and the Klenow fragment of DNA polymerase, as
previously described.33 Samples (15 µg of whole-cell
extract or 7 µg of nuclear extract) were preincubated with 2 µg of
poly dI-dC for 20 to 30 minutes and then the probe was
added and reaction mixtures were incubated on ice for an additional 20 minutes. Samples were then run on native 6% polyacrylamide gels in 0.5 × tris/borate/EDTA (TBE) buffer.
Histology of intestinal tissue.
Whole intestines were dissected from control or bone marrow
transplanted mice and fixed in 10% buffered formalin. Ten-micrometer paraffin sections were stained with hematoxylin and eosin and evaluated
by standard light microscopy. Intestinal IEL were identified as small
cells with densely staining nuclei that are located above the
epithelial basement membrane and below the plane of the epithelial cell
nuclei. Goblet cells, the nuclei of which are in the same location as
IEL, were distinguished from IEL by the presence of mucin vacuoles.
Sections from different mice were evaluated blindly and 2 experiments
were performed with similar results.
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RESULTS |
As a step toward evaluating possible gene therapy for XSCID, we
assessed whether a human c-expressing retrovirus could
correct the immunological defects in c-deficient mice.
Previously, it was established that murine c can
functionally cooperate with human IL-2R in mediating responses to
human IL-2.34,35 Conversely, human c can
functionally cooperate with murine IL-4R in mediating responses to
murine IL-4.7 However, the ability of human
c to cooperate with the other relevant cytokine receptor
chains in response to murine c-dependent cytokines has
not been reported. Because the T-cell defect in
c-deficient mice is believed to result from defective
IL-7 signaling, we investigated the ability of human c
to cooperate with murine IL-7R in response to murine IL-7. We
transfected murine IL-7R together with human or murine c into 293T cells along with Jak3, Stat5a, and Stat5b to
create an in vitro reconstitution system similar to one previously
developed for IL-2 signaling.36 Cells were then analyzed
for the ability of murine IL-7 to induce Stat5 DNA binding activity as
evaluated by EMSAs. As shown in Fig 1, like
murine c (lane 4), human c cooperated
with murine IL-7R to mediate a response to murine IL-7 (lane 6).
This suggested that gene therapy using human c to
correct c deficiency in mice was a rational experimental
approach. Such an approach would potentially also determine the ability of human c to cooperate with murine
c-dependent cytokines in vivo and if successful would
allow the identical vector to be evaluated in murine and human cells.
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| Fig 1.
Human c can functionally cooperate with
murine IL-7R in mediating the response to murine IL-7. 293T cells
were transfected with expression vectors for Jak3, Stat5a, Stat5b, and
the following: murine IL-7R alone (lanes 1 and 2), murine IL-7R + murine c (lanes 3 and 4), or murine IL-7R + human c (lanes 5 and 6). Forty-eight hours later, they
were not stimulated (lanes 1, 3, and 5) or stimulated with 1 nmol/L
murine IL-7 (lanes 2, 4, and 6). Nuclear extracts were prepared and
EMSAs performed using the PRRIII probe corresponding to the IL-2
response element in the human IL-2R gene.37
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Therefore, we constructed a retroviral expression vector in which the
c cDNA was inserted into pMMP, a retroviral expression vector in which expression is under the control of the MPSV LTR. A
schematic is shown in Fig 2A. We
transfected Phoenix ecotropic retroviral packaging cells to make a
stable clone producing the virus (see Materials and Methods).
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| Fig 2.
pMMP-driven expression of c expressed in
peripheral blood of mice that received bone marrow transplantation of
human c-transduced c-deficient bone
marrow. (A) Schematic of pMMP-c. (B) Human
c was detected by PCR of genomic DNA from peripheral
blood cells from c-deficient mice that received
c-deficient bone marrow transduced with human
c, but not from wild-type mice or
c-deficient mice that received no treatment, bone marrow
from a wild-type mouse, or bone marrow from a mock-transduced
c-deficient mouse.
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Successful transduction of murine bone marrow cells with a retrovirus
directing expression of human c.
Bone marrow cells were harvested and treated as described in Materials
and Methods. These cells were infected by coculture with
virus-producing Phoenix cells, followed by continued exposure to viral
supernatant on plates coated with RetroNectin. As a control, bone
marrow cells were also cocultured with untransfected Phoenix cells
(mock transduction). Approximately 1 × 106
cells were injected intravenously into c-deficient
irradiated recipient mice, and the mice were returned to a standard
animal facility. Six to 10 weeks later, we analyzed
c-deficient mice and wild-type mice as controls, as well
as c-deficient mice that had received bone marrow from
wild-type mice or c-transduced or mock-transduced bone
marrow from c-deficient mice. As shown in Fig 2B, human
c DNA was detected in peripheral blood cells from the
c-deficient mice that received bone marrow transduced with the human c.
Reconstitution of T cells, B cells, and NK cells in
c-deficient mice.
Because of the greatly diminished numbers of T, B, and NK cells in
c-deficient mice,24-26 we evaluated the
extent to which cellularity of these lineages was normalized by gene
therapy. As shown in Fig 3A, the number of
thymocytes was markedly increased in the mice receiving the
c-transduced, but not in mice receiving the
mock-transduced, bone marrow. Similarly, although
c-deficient splenocytes exhibit a substantially
decreased percent of CD3+ T cells with an elevated CD4:CD8
ratio as compared with wild-type mice (Fig 3B and C, second v
first dot plots), both the percent of CD3+ T cells (Fig 3B)
and the CD4:CD8 ratio (Fig 3C) were substantially corrected in the mice
receiving c-transduced bone marrow (fourth dot plot in
each panel), but not in those receiving mock-transduced bone marrow
(fifth dot plot in each panel). There was also partial correction of NK
cells in the spleen (Fig 3D). Analysis of spleen (Fig 3B) and bone
marrow (Fig 3E) in the c-transduced mice also showed a
substantial correction of the number of B cells. The numbers of
reconstituted T cells, B cells, and NK cells in the spleen are shown in
Table 1. Because c-deficient
mice exhibit a peripheral expansion of CD4+ T
cells,25,37 it is only after irradiation that this
population is diminished (in Table 1, compare mock-transduced to
nontransduced mice). Thus, the reconstitution in
c-transduced mice is much better than that seen in the
mock-transduced recipients and approximately equivalent to that seen in
mice receiving wild-type bone marrow.
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| Fig 3.
Successful reconstitution of T cells, B cells, and NK
cells in c-deficient mice by a gene therapy approach.
(A) Increased thymocytes after reconstitution of
c-deficient mice with c-deficient bone
marrow that was transduced with human c. Note that the
ordinate is a log scale. (B and C) Increased splenic T cells (B) with
normalization of the CD4:CD8 ratio (C) in c-deficient
mice with c-deficient bone marrow that was transduced
with human c; (B) also indicates correction of the
B-cell defect in splenocytes. (D) Increased numbers of splenic
TCRNK1.1+ cells after reconstitution of
c-deficient mice with c-deficient bone
marrow that was transduced with human c. Note that the
TCR+NK1.1 cells are partially diagonally
shifted to the right (albeit still to the left of the
NK1.1+ cells that are boxed in each dot plot); this is an
artifact that resulted from the use of 4-color flow cytometry. (E)
Increased B cells in bone marrow after reconstitution of
c-deficient mice with c-deficient bone
marrow that was transduced with human c. In (B through
E), we have selected representative dot plots for each of the cell
types analyzed from our final 3 different gene therapy experiments in
which all analyses were performed. Our initial experiments provided
consistent information, but not all assays were included in those
experiments. In the final 3 experiments, splenic cellularities (mean ± standard error of mean [SEM] × 106) were as
follows: wild-type mice (87.0 ± 3.0, top dot plots),
c-deficient mice (49.0 ± 5.5, second dot plots),
c-deficient mice, which received wild-type bone marrow
(77.8 ± 19.9, third dot plots), c-deficient mice,
which received human c-transduced bone marrow (82.8 ± 15.6, fourth dot plots), and c-deficient mice, which
received mock-transduced bone marrow (20.8 ± 3.9, bottom dot plots).
The data in (A) were from the final 2 experiments and those in (B)
through (E) were representative of the final 3 experiments. The number
of mice analyzed in each experiment was as follows: experiment 1 (mice
analyzed at 63 days after bone marrow transplantation): 1 wild-type
mouse, 2 c KO mice (no treatment), 2 c KO
mice (WT-BMT), 3 c KO mice
(hc-transduced), and 1 c KO mouse
(mock-transduced). Experiment 2 (mice analyzed at 62 days after bone
marrow transplantation): 2 wild-type mice, 2 c KO mice
(no treatment), 1 c KO mouse (WT-BMT), 2 c KO mice (hc-transduced), and 1 c KO mouse (mock-transduced). Experiment 3 (mice
analyzed at 52 days after bone marrow transplantation): 1 wild-type
mouse, 2 c KO mice (no treatment), 1 c KO
mouse (WT-BMT), 3 c KO mice
(hc-transduced), and 2 c KO mice
(mock-transduced). Experiment 3 was not included in (A) because
thymocyte numbers were not counted.
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Whereas splenocytes from c-deficient mice transplanted
with mock-transduced bone marrow did not exhibit native and
IL-2-induced lytic activity against the NK-sensitive target cell,
YAC-1, splenocytes from c-transduced mice had detectable
cytolytic activity, suggesting that the NK cells reconstituted in these
mice were functional (data not shown). The reconstituted B cells were
also functional as evaluated by the augmentation of IgA, IgG, and IgM
Ig levels associated with reconstitution
(Table 2). In addition to correction of
lymphoid populations in the thymus, spleen, and bone marrow, transduction of c allowed the reconstitution of
intraepithelial lymphocytes in the intestine
(Fig 4). Consistent with the reconstitution of these lymphocyte populations, we confirmed expression of human c in thymocytes, splenocytes, and bone marrow cells, as
evaluated by flow cytometry using TUGh4 MoAb to human c
(PharMingen) (compare Fig 5A, C, and E to
Fig 5B, D, and F). Note that in the thymus, it is evident that the
entire peak is shifted, suggesting that most or all of the thymocytes
express human c. In the spleen, c was
expressed on CD3+B220,
CD3B220+, and
CD3B220 cells. In the bone
marrow, both B220+ and B220 populations
expressed c (data not shown). Based on forward versus side scatter, the lymphoid cells in the bone marrow expressed more
c than did the myeloid cells (data not shown).
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| Fig 4.
Reconstitution of IEL in c-deficient mice
that received c-transduced bone marrow. Paraffin
sections of small intestinal tissue were stained with hematoxylin and
eosin and evaluated for the presence of IEL by light microscopy
(40×). wt, wild-type mouse; cKO,
c-deficient mouse that received no treatment;
cKO-mock, c-deficient mouse that received
mock-transduced c-deficient bone marrow;
cKO-wt, c-deficient mouse that received
wild-type bone marrow; cKO-hc,
c-deficient mouse that received
c-deficient bone marrow transduced with human
c. Sections shown are representative of results from 2 separate experiments. Arrows indicate IEL in the wt,
cKO-wt, and cKO-hc mice.
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| Fig 5.
Expression of human c protein in
c-transduced, but not mock-transduced mice. Human
c was expressed on the surface of thymocytes (A and B),
splenocytes (C and D), and bone marrow cells (E and F). Shown are
representative mice from 1 of 5 experiments. Cells were stained with
TUGh4 rat anti-human c (PharMingen) or a rat IgG2b
isotype control.
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Partial correction of the CD4:CD8 T-cell ratio and IL-2-induced
signaling by retroviral transduction of human c in
c-deficient mice.
As previously reported, splenic T cells from
c-deficient mice have an activated memory phenotype,
with an increase in CD62Llow cells.28 However,
after retroviral transduction with the c retrovirus, the
wild-type naive pattern was restored (Fig
6A). Moreover, anti-CD3 stimulation potently induced IL-2R (CD25) expression on the c-transduced, but not mock-transduced
CD4+ T cells (Fig 6B). As shown in
Fig 7, we also evaluated the ability of the
splenocytes from variously treated mice to respond to IL-2. Splenocytes
from wild-type mice exhibited the greatest relative increase in
proliferation in response to IL-2 and cells from
c-deficient mice receiving mock-transduced
c-deficient bone marrow did not proliferate in response
to IL-2. Interestingly, splenocytes from mice receiving either
c-transduced or wild-type bone marrow proliferated to a
similar extent. The absolute increase in proliferation was as high as
seen with wild-type mice in 5 of 8 c-transduced mice examined, but the relative increase was lower due to the very low basal
proliferation in wild-type mice. The reason for the differences in
basal proliferation levels is unclear. Note that splenocytes from
c-deficient mice (Fig 7B) have a similarly high basal
proliferation in response to IL-2 as compared with splenocytes from
c-deficient mice receiving any type of bone marrow
transplant (Fig 7C, D, and E). Finally, as shown in
Fig 8, splenocytes from mice that received
c-transduced bone marrow could respond to IL-2
effectively, as judged by Stat5 DNA binding activity.
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| Fig 6.
Normalization of T-cell activation markers after gene
therapy of c-deficient mice. (A) CD4+ T
cells from c-deficient mice transduced with human
c exhibited normalization of CD62L expression. (B)
CD4+ T cells from c-transduced mice
exhibited normalization of anti-CD3-induced IL-2R expression,
whereas cells from mock-transduced mice did not. The specificity of
this finding was confirmed by the use of an isotype-matched antibody
(data not shown). Shown are data for wild-type and
c-deficient mice or c-deficient mice that
receive c-deficient bone marrow mock-transduced or
transduced with human c. Shown are representative mice
from 1 of 5 experiments.
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| Fig 7.
c-transduced mice exhibited IL-2-induced
proliferation of fresh splenocytes, whereas mock-transduced mice did
not. 3H-thymidine incorporation assays were performed as
described in Materials and Methods for wild-type mice (A),
c-deficient mice (B), or in c-deficient
mice that received a bone marrow transplant from wild-type mice (C) or
c-deficient bone marrow that was transduced with human
c (D) or mock-transduced (E). Shown are pooled data from
3 experiments; each pair of connected points represents an individual
mouse.
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|
View larger version (31K):
[in this window]
[in a new window]
| Fig 8.
c-transduced mice exhibited IL-2-induced
STAT DNA binding activity. Whole-cell extracts were prepared from
splenocytes that were cultured without or with 2 nmol/L IL-2 for 15 minutes, and EMSAs were performed using the -casein probe.
|
|
In view of our successful transduction of murine bone marrow, we also
prepared an amphotropic version of the pMMP-human c virus. As expected based on previous studies,16-18 this
virus could successfully direct the expression of human
c in an EBV-transformed B-cell line derived from a
patient with XSCID and moreover could confer to these cells the ability
to mediate IL-2-induced and IL-4-induced STAT protein activation
(data not shown).
| |
DISCUSSION |
XSCID is a severe inherited disease that is uniformly fatal when bone
marrow transplantation is not successful. In this study, we used
c-deficient mice as a model for human XSCID and tested whether a retroviral-mediated gene therapeutic approach could correct
in vivo the defects in these mice. Whereas no correction was detected
in mice that received mock-transduced c-deficient bone
marrow, substantial correction of lymphoid development of T cells, NK
cells, and B cells was observed in mice receiving c-transduced bone marrow. The T cells not only increased
in number, but their cell surface phenotypic profiles also normalized.
Specifically, the CD4:CD8 ratio largely was corrected, and resting T
cells no longer exhibited a memory-activated phenotype (increased CD62L expression). Moreover, T-cell function was partially restored as
demonstrated by responsiveness to IL-2. In addition, intestinal intraepithelial lymphocytes were reconstituted. B-cell function also
improved as evaluated by the increase in serum Ig levels. Importantly,
the retrovirally transduced bone marrow was almost as effective as
wild-type bone marrow. As such, these studies demonstrate functional
reconstitution in a murine in vivo model of XSCID. These studies
complement a study in which mice deficient in Jak3 were treated by a
similar gene therapy type of approach.21 Thus, for both of
the two major forms of
TB+NK SCID, gene
therapy has been tested and demonstrated to be effective in murine
models. Although we have demonstrated that reconstitution occurred, the
latest time point at which we analyzed mice in this study was 63 days
postbone marrow transplantation. Thus, it is unknown whether life-long
reconstitution was achieved, and it is possible that the number of
transduced clones might decrease with time. Nevertheless, it is
relevant that approximately 80% of lymphocyte engraftment typically
occurs within 2 months after bone marrow transplantation.38
Moreover, it is reasonable to hypothesize that
c-transduced cells might exhibit a growth advantage and/or augmented survival given their ability to respond to
c-dependent cytokines, which are well-known to induce
both proliferative and antiapoptotic signals.39
The fact that the retroviral transduction of human c
effectively restored T- and NK-cell development, as well as IL-2
signaling in mice, indicates that human c successfully
cooperated with murine IL-2R (which is essential for IL-2-dependent
proliferation and IL-15-dependent development of NK cells) and with
murine IL-7R (which is essential for IL-7-dependent development of
T cells and B cells in mice) in response to the endogenous murine
cytokines. The effectiveness of human c virus in murine
stem cell transduction also suggested that an amphotropic version of
the virus used in this study might have potential use for human gene
therapy. As a preliminary step in this direction, we confirmed that
such a virus could successfully transduce an EBV-transformed B-cell
line from a patient with XSCID and confer de novo responsiveness to IL-2 and IL-4.
In XSCID, haploidentical bone marrow transplantation is highly
successful even though many individuals do not successfully engraft B
cells. Thus, in an investigational gene therapy clinical protocol, one
would want to assure that conventional therapy was still available even
if gene therapy were not successful. In this regard, it is appealing to
contemplate transduction of cord blood stem cells in the perinatal
period, allowing ample time for conventional bone marrow
transplantation should gene therapy be unsuccessful. As haploidentical
bone marrow transplantation is not uniformly successful, gene therapy
is obviously a desirable eventual approach for the treatment of XSCID.
| |
ACKNOWLEDGMENT |
We thank Drs Jeng-Shin Lee and Richard C. Mulligan, Harvard Medical
School, for providing the pMMP vector; Dr Garry Nolan, Stanford
University, for providing Phoenix cells; Dr Harry Malech and Gilda
Linton, NIAID, for providing 293 SPA cells; Dr Jennifer Puck, NHGRI,
for providing the human XSCID EBV line mentioned in the results; and Dr
Atsushi Miyajima for the pME18S expression vector; and Drs Cynthia
Dunbar and Harry Malech for valuable conversations/critical comments.
| |
FOOTNOTES |
Submitted February 11, 1999; accepted July 8, 1999.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Warren J. Leonard, MD, Bldg
10, Room 7N252, Lab of Molecular Immunology, NHLBI, NIH, Bethesda, MD
20892-1674; e-mail: wjl{at}helix.nih.gov.
| |
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ABSTRACT
INTRODUCTION