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Blood, 15 March 2001, Vol. 97, No. 6, pp. 1618-1624
GENE THERAPY
Lack of dominant-negative effects of a truncated  c on
retroviral-mediated gene correction of immunodeficient
mice
Makoto Otsu,
Kazuo Sugamura, and
Fabio Candotti
From the Clinical Gene Therapy Branch, National
Human Genome Research Institute, National Institutes of Health,
Bethesda, MD; and the Department of Microbiology and Immunology,
Tohoku University School of Medicine, Sendai, Japan.
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Abstract |
A recent clinical trial of gene therapy for X-linked severe
combined immunodeficiency (XSCID) has shown that retroviral-mediated gene correction of bone marrow stem cells can lead to the development of normal immune function. These exciting results have been preceded by
successful immune reconstitution in several XSCID mouse models, all
carrying null mutations of the common gamma chain ( c). One question
not formally addressed by these previous studies is that of possible
dominant-negative effects of the endogenous mutant c protein on the
activity of the wild-type transferred gene product. The present work
was therefore undertaken to study whether corrective gene transfer was
applicable to an XSCID murine model with preserved expression of a
truncated c molecule ( c+-XSCID). Gene correction
of c+-XSCID mice resulted in the reconstitution of
lymphoid development, and preferential repopulation of lymphoid organs
by gene-corrected cells demonstrated the selective advantage of
c-expressing cells in vivo. Newly developed B cells showed
normalization of lipopolysaccharide-mediated proliferation and
interleukin-4 (IL-4)-induced immunoglobulin G1 isotype switching.
Splenic T cells and thymocytes of treated animals proliferated normally
to mitogens and responded to the addition of IL-2, IL-4, and IL-7,
indicating functional reconstitution of c-sharing receptors.
Repopulated thymi showed a clear increase of
CD4 /CD8 and CD8+
fractions, both dramatically reduced in untreated
c+-XSCID mice. These improvements were associated
with the restoration of Bcl-2 expression levels and enhanced cell
survival. These data indicate that residual expression of the
endogenous truncated c did not lead to dominant-negative effects in
this murine model and suggest that patient selection may not be
strictly necessary for gene therapy of XSCID.
(Blood. 2001;97:1618-1624)
© 2001 by The American Society of Hematology.
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Introduction |
X-linked severe combined immunodeficiency (XSCID),
the most common form of severe combined immunodeficiency, is
characterized by profound defects of humoral and cellular
immunity.1 Affected boys succumb during infancy to severe
infections, unless allogeneic bone marrow transplantation (BMT) is
successfully performed. Genetic defects of expression and function of
the c of cytokine receptors have been shown to be responsible for
this disease.2,3 The involvement of c in multiple
cytokine receptors, including those for interleukin-2 (IL-2), IL-4,
IL-7, IL-9, and IL-15, explains the severe impairment of lymphoid
development and function in XSCID patients,4,5 as has also
been illustrated by the generation of a series of c-knockout
mice,6-9 which manifest a severe immunodeficient phenotype.
Since its first application in 1968,10 BMT has been
performed as a treatment for XSCID with great
success.11,12 However, potential severe complications,
such as graft-versus-host disease, and incomplete reconstitution of
B-cell immunity make BMT imperfect and leave room for improvement.
Genetic correction of autologous hematopoietic stem cells (HSCs) has
been proposed as a beneficial alternative therapeutic approach for this
disease.13-17
Recently, we and others have demonstrated the feasibility of stem cell
gene therapy for XSCID using murine models.18-20 Soon thereafter, the results of the first human clinical trial for XSCID
patients were reported and showed clear evidence of a clinical benefit
associated with the gene correction procedures.21
Mutated proteins have been demonstrated to act as transdominant
inhibitors of wild-type protein functions in various
systems,22,23 and dominant-negative effects of endogenous
mutant proteins may hinder the efficacy of gene therapy
attempts.23 Mutation analysis efforts have shown that a
significant number of XSCID patients carry genetic aberrations
compatible with residual c protein expression.24 These
mutant receptor chains may show dominant-negative effects against the
wild-type c introduced in the patients' cells by gene transduction,
thus limiting the efficacy and potential applications of gene therapy
for XSCID. We have used a murine model of XSCID carrying a truncated
c to formally examine whether retroviral-mediated gene therapy is
affected by the presence of residual endogenous c expression.
Treated mice showed restored lymphoid development with evidence of
coexpression of mutant and ectopic normal c, suggesting that the
presence of the truncated c did not hinder the therapeutic effects
of gene transfer. Moreover, we demonstrated that the functional
reconstitution of c-mediated signaling led to a correction of
immunoglobulin (Ig) isotype switching in B cells and to the
up-regulation of Bcl-2 and enhanced cell survival in gene-corrected thymocytes.
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Materials and methods |
Mice
Generation and characterization of c+-XSCID
mice were previously described.8 As a result of gene
targeting, these mice express a truncated c protein with conserved
extracellular and transmembrane domains. We used affected male mice and
normal littermates that had been backcrossed onto a C57BL/6 background
for at least 15 generations. All animal experiments were approved by
the National Human Genome Research Institute Animal Care and
Use Committee.
Retroviral-mediated stem cell gene correction procedures
The retroviral producing cell line and transduction procedures
were previously described.19 Briefly, the retroviral
vector MNDmc, carrying the murine c (mc) cDNA, was constructed
using the MND-X-IRES-EGFP vector (gift of Dr D. B. Kohn,
Children's Hospital, Los Angeles, CA) after removal of the IRES-EGFP
fragment and packaged using the ecotropic GP+E-86 cells.25
Bone marrow (BM) cells obtained from 5-fluorouracil (5-FU)-treated
c+-XSCID mice (4-14 weeks old) were prestimulated for
48 hours with 10 ng/mL murine IL-3 (Peprotech, Rocky Hill, NJ), 100 ng/mL human IL-6 (Amgen, Thousand Oaks, CA), and 100 ng/mL rat stem
cell factor (Amgen). After 48 hours of co-cultivation with producing
cells, 1 to 4 × 106 nonadherent cells were injected
intravenously into lethally irradiated (9 Gy) 8-week-old
c+-XSCID mice (referred to as mc-BMT mice). As
control, BM cells harvested from either normal or
c+-XSCID mice after 5-FU treatment were mock
transduced and transplanted into c+-XSCID mice. The
animals receiving normal and c+-XSCID BM cells were
designated control-BMT and mock-BMT mice, respectively.
Polymerase chain reaction-based estimation of transgene
copy numbers
The polymerase chain reaction (PCR) method used to assess
transgene copy numbers in vector-transduced cells was previously described.19 Briefly, genomic DNA was prepared from tissue
samples using the SNAP genomic DNA isolation kit
(Invitrogen, Carlsbad, CA). The integrated provirus and  -globin
sequences were co-amplified for 29 and 25 cycles, respectively, from 20 ng test DNA and reference standards. Amplified products were then
analyzed using the BIT Image software (Aladdin Systems, Watsonville,
CA) and the NIH Image software (http://rsb.info.nih.gov/nih-image) as
described.19
Reverse transcription-polymerase chain reaction analysis of
c expression
Total RNA samples were prepared from BM cells with the QIAamp
RNA Blood Mini Kit (Qiagen, Valencia, CA), followed by first-strand DNA
synthesis using the Retroscript kit (Ambion, Austin, TX). The following
primers were used for PCR amplification of obtained cDNA: P1,
5'-CTGCTCAGAATGCCTCCAATTCC-3'; P2, 5'-CCTGCGTGCAATCCATCTTGTTCAAT-3'; P3, 5'-TCTGCAGCCAGACTACAGTG-3'; P4, 5'-GATCCAGATTGCCAAGGTGAGTAG-3'; P5,
5'-GCTCGTACTCTATAGGCTTC-3'. The cyclophilin primers were from the
QuantumRNA kit (Ambion). Equal amounts of first-strand cDNA were added to a mixture of 0.2 µM dNTP, 1.5 mM MgCl2, 2.5 U AmpliTaq DNA polymerase, 1× PCR buffer (PE Biosystems, Foster City,
CA), and 0.4 µM each primer. PCR reactions were performed using 34 amplification cycles (94°C for 30 seconds, 58°C for 30 seconds), and the amplified products were separated on a 2% agarose gel and then
visualized by ultraviolet light.
Flow cytometry analysis of peripheral blood lymphocytes
Peripheral blood (PB) samples were collected from retro-orbital
sinus 8 weeks after BMT, and white blood cells were enumerated using a
Multisizer (Coulter Electronics, Hialeah, FL). PB samples were stained
with combinations of monoclonal antibodies (mAbs) described below. The
percentage of each lymphoid fraction was determined by flow cytometry
using a FACSCalibur and the CellQuest software (Becton Dickinson, San
Jose, CA), and the obtained value was used to estimate the absolute
cell count of each PB lymphocyte subset. The following mAbs were used:
fluorescein isothiocyanate (FITC)-anti-CD8a (Ly-53-6.7), phycoerythrin
(PE)-anti-CD62L (L-selectin; MEL-14), CyChrome (Cy)-anti-CD4 (GK1.5),
PE-anti-IgMb (Igh-6b), Cy-anti-CD45R/B220 (RA3-6B2),
PE-anti-natural killer (NK) 1.1 (Ly-55), and Cy-anti-CD3e
(145-2C11). All flow cytometry reagents were purchased from Pharmingen
(San Diego, CA).
In vitro immunoglobulin isotype switching assay
Splenocytes obtained from euthanized mice at 18 to 22 weeks
after BMT were plated at 1 × 106 cells/mL in R-10 medium
(RPMI 1640 containing 10% fetal bovine serum, and 50 µM
2-mercaptoethanol). For the induction of IgG3 isotype switching,
lipopolysaccharide (LPS; 20 µg/mL; Sigma, St Louis, MO) was added to
the cultures. To induce IgG1 or IgE isotype switching, LPS plus IL-4
(25 ng/mL; Peprotech) were used with daily addition of 500 ng/mL
anti-interferon neutralizing Ab (R4-6A2) or 500 ng/mL rat IgG1
(R3-34). After 6 days of culture, cells were collected and stained with
either biotin-conjugated anti-IgG1 (A85-1), anti-IgG3 (R40-82),
anti-IgE (R35-72), or rat IgG1. Cells were then stained with Cy-B220
and streptavidin-PE, and analyzed by flow cytometry. All flow cytometry
reagents were from Pharmingen.
Cell proliferation assay
Splenocytes obtained from euthanized mice at 18 to 22 weeks
after BMT were seeded in triplicate in 96-well flat-bottom plates (1 × 105 cells/well) in R-10 medium. Thymocytes were
cultured in the same medium at 5 × 104 cells/well in
96-well U-bottom plates. Cells were cultured for 48 hours with or
without the addition of the mitogens listed below and were pulsed with
0.5 µCi/well of [3H]-thymidine (NEN-Dupont, Boston, MA)
for the final 16 hours. Cells were then harvested, and incorporated
radioactivity was determined by using a scintillation counter. The
stimulants used were as follows: LPS (20 µg/mL; Sigma), phorbol
myristate acetate (PMA; 10 ng/mL; Sigma), ionomycin (500 ng/mL;
Sigma), soluble anti-CD3e (145-2C11; 20 µg/mL; Pharmingen), soluble
anti-CD28 (37.51; 2 µg/mL; Pharmingen), concanavalin A (ConA; 2 µg/mL; Sigma), human IL-2 (500 U/mL), murine IL-4 (50 ng/mL; R&D
Systems, Minneapolis, MN), and murine IL-7 (50 ng/mL; Peprotech).
Immunophenotypic and functional analyses of thymocytes
Thymocytes were collected from euthanized animals at 18 to 22 weeks after BMT and then analyzed by flow cytometry. For
immunophenotype determination, cells were stained with FITC-CD8a and
Cy-CD4 mAbs. To assess mc expression, cells were stained with either
purified rat IgG2a (R35-95) or anti-mc mAbs [4G3 or
TUGm3],26 followed by biotin-antirat immunoglobulin and
streptavidin-PE. Bcl-2 expression levels were determined by
intracellular staining according to previously described
procedures27 with minor modifications. Briefly, cells were
first stained with FITC-CD8a and Cy-CD4 mAbs, then permeabilized using
the Cytofix/Cytoperm kit. This was followed by intracellular staining
with PE-isotype control or PE-anti-Bcl-2 mAb 3F11. To assess the
spontaneous death of thymocyte subsets, cells were cultured for 24 hours in R-10 medium, then stained with FITC-CD8a and PE-CD4 mAbs and
followed by the addition of 7-amino-actinomycin D (7-AAD). Dead cells
were defined as 7-AAD-positive cells in each fraction determined by
CD4/CD8 staining. All reagents, except for TUGm3, were purchased
from Pharmingen.
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Results |
Successful mc gene transfer and expression in hematopoietic stem
cells of c+-XSCID mice
BM cells collected from c+-XSCID mice were
transduced with the retroviral vector MNDmc and then infused into
irradiated recipient animals. At 8 weeks after BMT, 8 mc-BMT mice, 3 control-BMT mice, and 4 mock-BMT mice were available for analysis.
To assess the mc transgene expression in treated mice, we performed
reverse transcription (RT)-PCR analysis on BM samples. According to
the particular experimental group, mice were expected to express one or
more c transcripts (Figure 1A).
Amplification reactions using the P1 and P2 primers specific for the
targeted il-2rg allele (knockout allele) generated similar
levels of amplicons in samples from mc-BMT and mock-BMT mice (Figure
1B, lanes 5 and 7), whereas no product was obtained in untreated normal
mice (lane 3). Using the primers specific for exon 8 that has been targeted in c+-XSCID mice (P3 + P4), amplicons
were generated only in samples from untreated normal mice reflecting
endogenous c-mRNA and mc-BMT mice representing retroviral
vector-mediated transgene expression (Figure 1B, lanes 3 and 5). When
amplified with vector-specific primers (P3 + P5), c transcripts
were demonstrable only in the sample originated from mc-BMT mice
(Figure 1B, lane 5), confirming retroviral vector-mediated transgene
expression in treated mice. Similar intensity of internal control
cyclophilin signals in all samples (lanes 3, 5, 7) and absence
of amplification products in RT minus reactions (lanes 2, 4, 6)
confirmed the accuracy of this study. Collectively, retroviral-mediated
c gene transfer into BM cells of c+-XSCID mice
resulted in significant levels of expression of the ectopic, normal
c while in the presence of the truncation mutant c gene. Because
the truncated c is expressed on the surface of
c+-XSCID cells8, flow cytometry analysis
of c expression could not be easily used in this model as a measure
of successful gene transfer.
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| Figure 1.
Analysis of retroviral-mediated expression of c.
(A) Schematic representation of the relative location of primers used
for RT-PCR analysis of mc expression. Primers P1 and P2 are specific
for the transcripts derived from the targeted il-2rg allele
in c+-XSCID mice (knockout allele, KO). Primers P3
and P4 amplify equal fragments from the endogenous wild-type (WT
allele) and retroviral-expressed mc (MNDmc). Primers P3 and P5
were used to specifically amplify the MNDmc-derived transcripts. ex,
exon; neo, neomycin-resistant gene cassette. (B) RT-PCR analysis of
mc transcripts. Expression levels of c transcripts originated by
RT-PCR amplification of BM samples of wild-type c+,
gene-corrected (mc-BMT), and mock-treated (mock-BMT) mice are shown.
Amplicons were not detected in control samples without template
(NTC, lane 1) or in samples amplified in the absence of the reverse
transcriptase step (RT ). Similar amplification of cyclophilin
sequences demonstrated equal loading of each sample.
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Restored lymphocyte development by stem cell gene
correction
To assess whether c transgene expression in the presence of
mutant c leads to the restoration of lymphoid development in c+-XSCID mice, we analyzed PB samples at 8 weeks
after BMT. Absolute PB lymphocyte counts showed an approximately 8-fold
increase in mc-BMT mice compared to untreated
c+-XSCID mice, reaching levels comparable to those of
normal mice (Figure 2A, PBLs). FACS
analysis of PB lymphocytes demonstrated the reconstitution of all
lymphoid fractions, including mature B cells
(B220+/IgM+), naive CD4+ T cells,
CD8+ T cells, and mature NK cells
(CD3/NK1.1bright) (Figure 2A and data not
shown). Mature B cells increased by approximately 130-fold, though the
absolute values were still significantly lower than normal.
CD4+ T cells showed significant increases in total and
naive (CD62L+) cell counts (approximately 14- and 60-fold,
respectively), and CD8+ T cells increased by approximately
70-fold. No significant changes in any lymphocyte compartment were
observed in mock-BMT control mice (Figure 2A, mock), indicating that
the BMT procedure itself had no positive effects on lymphoid
reconstitution in transplanted animals. NK cells appeared in 7 of 8 treated mice at frequencies ranging between 0.1% and 0.9% of lymphoid
cells, whereas none of the untreated c+-XSCID or
mock-BMT mice showed detectable NK1.1+ cells (data not
shown). Altogether, retroviral-mediated mc gene transfer into HSCs
resulted in the efficient restoration of lymphoid development in
transplanted c+-XSCID animals.
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| Figure 2.
Assessment of lymphoid development and transgene copy
numbers in treated animals.
(A) Lymphoid development after stem cell gene therapy (8 weeks after
BMT). Presented are absolute counts of PB lymphocytes (PBLs), mature B
cells (B220+/IgM+), CD4+ T cells
(total and CD62L+), and CD8+ T cells. Shown are
the mean (±SD) values obtained from 6 wild-type mice
(c+), 9 untreated c+-XSCID mice
(XSCID), 4 mock-BMT mice (mock), 8 mc-BMT mice (mc), and 3 control-BMT mice (control). (B) Transgene copy numbers in mouse
tissues. PCR products derived from the mc-provirus (326 bp) and
control -globin sequences (401 bp) were separated on agarose gel and
quantified as described in "Materials and methods." Copy numbers
estimated by interpolation with the reference standard are indicated.
Sp; splenocytes, Thy; thymocytes. As expected, MNDmc-bands are
absent in BM samples obtained from untreated c+-XSCID
mice (XSCID, lane 2) and mock-BMT mice (mock, lane 3).
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Copy number assessment of mc transgene in
treated mice
We next set out to quantify the mc transgene in
lymphohematopoietic tissues obtained from treated animals. As shown in
Figure 2B, transgene-specific signals were detected in BM, spleen, and thymus of mc-BMT mice (lanes 4-6), but not in BM samples obtained from untreated c+-XSCID or mock-BMT mice (lanes 2-3).
Estimation of average transgene copy numbers demonstrated that the BM
samples had relatively low copies of the provirus (approximately 0.06 copies/cell), whereas peripheral lymphoid tissues contained much higher
copy numbers of the mc-transgene (spleen, approximately 0.35;
thymus, more than 0.5 copies/cell). Analysis of a second mc-BMT
mouse led to similar results (BM, approximately 0.10; spleen,
approximately 0.38; thymus, more than 0.5 copies/cell; data not shown).
These results indicate that although relatively low numbers of
transduced HSCs engrafted in the BM, gene-corrected cells accumulated
and repopulated peripheral lymphoid tissues, especially the thymus, suggesting a selective advantage over noncorrected cells.
Assessment of immunoglobulin isotype switching in B
lymphocytes
We and others have previously demonstrated that
retroviral-mediated gene correction of XSCID mice results in normal
immunoglobulin levels and in the development of humoral immune
responses to foreign antigens.18-20 To confirm and extend
these findings, we performed in vitro immunoglobulin isotype switching
assay on splenic B lymphocytes. DiSanto et al6 have shown
that splenic B cells obtained from c-deficient mice were able to
produce IgG3 on LPS stimulation but failed to switch to IgG1-producing
cells in response to LPS plus IL-4, indicating an indispensable role of
c in IL-4-mediated immunoglobulin isotype switching. Consistent
with this observation, we found that LPS stimulation induced IgG3
surface-positive B cells regardless of the presence or the absence of
normal c (Figure 3A). In contrast, the
addition of IL-4 to LPS-stimulated B cells resulted in the appearance
of IgG1 surface-positive cells in wild-type (c+)
and mc-BMT animals, but not in untreated c+-XSCID
mice (XSCID), suggesting that transduced c allowed the IL-4-mediated signaling required for IgG1 class switching (Figure 3B).
Similar experiments assessing IgE class switching demonstrated that IgE
surface-positive cells could be induced only in B cells from wild-type
mice and c+-XSCID mice after gene correction (data
not shown). We conclude that stem cell gene correction of
c+-XSCID mice resulted in the reconstitution of IL-4
receptor-mediated signaling and the restoration of proper
immunoglobulin isotype switching.
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| Figure 3.
Analysis of in vitro immunoglobulin isotype switching in
splenic B cells.
(A) LPS stimulation induced IgG3 expression on B220+ B
cells obtained from normal c+, untreated
c+-XSCID (XSCID), and gene-corrected
c+-XSCID (mc-BMT) mice. (B) LPS alone failed to
induce IgG1 expression, whereas in combination with IL-4,
IgG1-expressing B220+ cells developed in samples from
normal and mc-BMT mice but not from untreated
c+-XSCID mice. Shown are representative results of 2 independent experiments.
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Proliferative responses of lymphocytes
We next examined whether lymphoid cells developed in mc-BMT
mice proliferated in response to mitogens. When stimulated with LPS,
splenocytes obtained from treated animals proliferated similarly to
normal splenocytes, whereas cells from untreated
c+-XSCID mice showed extremely poor responses (Table
1). Proliferation of T cells was tested
by stimulating splenocytes with PMA plus ionomycin or plate-bound
anti-CD3 plus soluble anti-CD28 mAbs. As shown in Table 1, splenocytes
of untreated c+-XSCID mice showed only marginal
responses, whereas splenocytes of mc-BMT mice exhibited significant
proliferative responses to both stimuli at levels comparable
to normal.
To examine whether the transduced c subunit could allow for cytokine
responses in T cells, we investigated thymocyte proliferation to ConA
stimulation with or without IL-2, IL-4, and IL-7. Consistent with
previous observations,6,8 thymocytes obtained from
untreated c+-XSCID animals did not show significant
proliferation in response to ConA, nor was the response enhanced by the
addition of cytokines (Table 2). In
contrast, thymocytes developed in mc-BMT mice showed ConA-mediated
proliferation at levels similar to normal, and the addition of
cytokines further increased cell proliferation, though to a lesser
degree than that observed in normal thymocytes (Table 2). These results
indicate the reconstitution of functional c-containing cytokine
receptors in T cells derived from treated animals.
Immunophenotypic analysis of thymocytes
Although the repopulation of thymus in XSCID mice after
retroviral-mediated gene therapy was suggested in a previous report by
the increased thymocyte numbers,18 detailed
immunophenotypic analysis was not reported. We deemed it important to
determine the extent of normalization of thymic subpopulations induced
in c+-XSCID mice by corrective gene transfer. We
assessed thymic cellularity and found a dramatic increase of thymocyte
counts (average, 3.7 × 107) in 3 of 6 treated animals,
whereas untreated c+-XSCID or mock-BMT mice showed
much lower counts ranging between 3 × 105 and
4 × 106. As expected, the expression of mc was
detected on thymocytes from all animals by flow cytometry (Figure
4, left panels). When stained for CD4 and
CD8, untreated c+-XSCID animals showed minimal
numbers of CD4/CD8 and CD8+
thymocytes, consistent with previous observations.28,29 In contrast, marked increases of CD4/CD8 and
CD8+ cells were observed in thymocytes obtained from
treated animals (Figure 4, right panels). Taking into account the
significant increase in thymocyte numbers observed in treated mice, the
improvement of CD4/CD8 and CD8+
cellularity appears even more significant.
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| Figure 4.
Immunophenotyping of thymocytes.
Thymocytes obtained from wild-type c+, untreated
c+-XSCID (XSCID), and gene-corrected
c+-XSCID animals (mc-BMT) were analyzed. Left
panels show mc expression on unfractionated thymocytes. Shown are
the staining profiles of isotypic control mAbs (gray histograms) and
mc-specific mAbs (open histograms). Right panels show CD4 versus CD8
staining of thymocytes. Percentages of cells in each quadrant are
shown. Results are representative of 2 independent experiments.
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Bcl-2 expression and cell survival in thymocytes
Recent studies have suggested that diminished Bcl-2 expression and
reduced cell survival may explain the poor thymic cellularity in XSCID
mice.28,29 To test whether restored c-mediated
signaling had positive effects on these parameters, we examined Bcl-2
levels and cell viability of thymocytes by flow cytometry. We confirmed the marked reduction of Bcl-2 expression, especially in
CD4/CD8 and CD8+ fractions of
thymocytes obtained from untreated c+-XSCID animals
(Figure 5A). In contrast, thymocytes
obtained from mc-BMT mice showed clear improvement of Bcl-2 levels
in all thymocyte fractions, particularly in
CD4/CD8 and CD8+ cells (Figure
5A). When cell viability was assessed in thymocytes kept in culture for
24 hours, extensive cell death was observed in
CD4/CD8 and CD8+ cells derived
from untreated c+-XSCID mice (Figure 5B, white bars)
compared to normal thymocytes (black bars). Consistent with the
enhanced Bcl-2 levels, thymocytes of mc-BMT animals showed clear
improvement of cell survival in these 2 fractions (Figure 5B, gray
bars). These results demonstrated that the retroviral-mediated gene
correction resulted in the reversion of the defects observed in
c+-XSCID thymocytes, most likely because of the
restoration of c-mediated signaling pathways (eg, IL-7) required for
normal thymocyte homeostasis in vivo.
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| Figure 5.
Enhanced Bcl-2 expression and cell survival after
stem cell gene correction.
(A) Bcl-2 expression levels in thymocyte fractions. Thymocytes were
stained for surface CD4 and CD8 expression, then stained
intracellularly with either isotypic control mAb (gray histograms) or
antimouse Bcl-2 mAb (open histograms). Shown are the results analyzed
by gating CD4/CD8 (DN) cells,
CD4+/CD8+ (DP), or either CD4+ or
CD8+ cells. Representative results of 2 independent
experiments are presented. (B) In vitro assessment of thymocyte cell
death. After 24 hours of culture, cells were stained for CD4 and CD8,
then incubated with 7-AAD. The percentage of 7-AAD+ cells
was determined in each thymocyte fraction by gating on
CD4/CD8, CD4+/CD8+,
CD4+, and CD8+ cells. Shown are the mean values
of 2 independent experiments.  , c+, untreated normal
mice;  , XSCID, untreated c+-XSCID mice;  ,
mc-BMT, c+-XSCID mice transplanted with
mc-transduced BM cells.
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| |
Discussion |
An extensive series of in vitro13-17 and in
vivo18-20 preclinical experiments performed by several
laboratories since the early 1990s has culminated in the recent
exciting results of the first clinical trial of gene therapy for XSCID
that has shown good immune reconstitution in treated patients for more
than 1 year.21 Important information on the safety and
efficacy of c gene transfer into HSCs was obtained using different
mouse strains of XSCID, all carrying null-mutations of the
c.18-20 However, whether the residual expression of
c could result in dominant-negative effects, thus potentially
reducing the therapeutic effects of gene transfer, still need to be
formally investigated.
We found that retroviral-mediated expression of the normal c
molecule into hematopoietic stem cells of mice carrying a truncated c resulted in the restoration of cellular and humoral immune functions in transplanted mice. In particular, thymic development and
mature T lymphocyte numbers and functions were reconstituted to levels
close to those observed in control animals. At partial contrast, total
numbers of peripheral B lymphocytes and NK cells detected in treated
animals were lower than normal. These findings are similar to those of
previously published reports18-20,30 and may indicate that
different levels of transgene expression may be needed for the complete
normalization of B- and NK-cell development. Regardless of the low
total numbers of B lymphocytes that developed in treated mice in these
previous studies, humoral immunity was clearly
improved.18-20,30 The reconstitution of cytokine
responses in the B cells of these animals, however, had not been
studied in detail. IL-4 is known to induce IgG1 and IgE isotype
switching in LPS-activated mouse B cells,31 and c is
thought to be indispensable for this response.6,32 To
examine IL-4 signaling in XSCID B cells after genetic correction, we
opted for an in vitro immunoglobulin isotype switching assay that
enables the functional assessment on a single-cell basis. We showed
that B cells newly developed in treated animals switched to
IgG1+ or IgE+ cells in response to IL-4,
whereas untreated c+-XSCID counterparts failed to do
so. These results indicate that the c expressed in B cells by gene
transfer is functionally competent in vivo and further support the
notion that humoral responses can be corrected in XSCID mice by gene
transfer. However, it is important to note that, because B
lymphopoiesis differs significantly in mice and humans and because of
the differences existing between the B-cell phenotype of the murine
model and of humans with XSCID, these results may not directly
translate to human trials.
We have paid particular attention to the analysis of thymocyte
populations in treated mice in terms of immunophenotype and function
because studies of these critical cells are obviously precluded in
humans treated by gene therapy. By assessing proliferative responses to
various stimulants, we could demonstrate that thymocytes developed in
treated animals responded to the addition of IL-2, IL-4, or IL-7,
indicating the functional reconstitution of the respective
c-containing receptors. The proliferative response, however, was not
as strong as that observed in normal thymocytes (Table 2). Soudais et
al20 have also reported that the enhancement of ConA
mitogenic responses by the addition of IL-2 or IL-7 in splenocytes
derived from gene therapy-treated XSCID animals was reduced compared
to that in normal counterparts. These partial responses may be
attributed to lower expression levels of transduced c compared to
normal or to the lack of physiological regulation of c expression,
which is under the constitutive transcriptional control of the
retroviral long terminal repeat. At present, it is unclear
whether higher levels of c expression would result in normal
performances of developing lymphocytes in XSCID recipients treated by
gene therapy or whether activation-mediated induction of c
expression is necessary to achieve full immune reconstitution. With the available technology, achievement of high levels of
transgene expression above a hypothetical threshold may be more
feasible than attempting the reconstitution of the physiological
mechanisms of c regulation.
By immunophenotyping thymocytes developed in mc-BMT mice, we
observed a dramatic increase of both
CD4/CD8 and CD8+ cells, the
thymic subsets markedly affected in c-deficiency mice.28,29 The higher Bcl-2 expression levels and the
concomitant improvement of cell survival observed in these fractions
are most likely attributable to the reconstitution of functional
c-containing receptors because c-dependent cytokines have been
shown to enhance lymphocyte cell survival by maintaining the levels of
antiapoptotic molecules including Bcl-2 and
Bcl-xL.33,34 Specifically, because reduced
Bcl-2 levels are observed in CD4/CD8
thymocytes of IL-7-deficient mice,35 signaling through
the reconstituted IL-7 receptors leading to the expansion of thymic precursors is primarily responsible for thymic reconstitution in our
treated animals, though new lines of evidence have recently challenged
the role of c-dependent cytokines in the up-regulation of Bcl-2
during positive selection.36
Analysis of our mice also showed that the copy numbers of integrated
MNDmc provirus clearly varied among the different tissues. Whereas
BM cells contained 0.06 to 0.1 copies/cell of the mc transgene,
splenocytes and thymocytes showed much higher proviral copy numbers,
indicating preferential repopulation of these peripheral lymphoid
tissues by the gene-corrected cells. In gene therapy experiments using
Jak3-deficient SCID mice, Bunting et al30 also observed
higher proviral copy numbers in lymphoid cells than in myeloid
lineages. These observations are in line with the results of our
competition experiments in murine XSCID BMT models37 and
with the overall clinical experience of allogeneic BMT in patients with
XSCID, and they further support the notion that after stem cell gene
therapy, gene-corrected cells have a selective growth advantage over
defective cells in the reconstitution of lymphoid compartments in
conditions affecting the c/Jak3-signaling pathway. It was especially
striking that the thymocytes in this study showed nearly one copy of
the mc transgene per cell, suggesting that most cells that
repopulated each recipient's thymus were gene-corrected. Several
explanations are possible for these findings, including the enhanced
ability of thymic precursors expressing c to home and engraft in
thymic tissues and the preferential survival of c-positive cells in
the thymic environment.
In summary, we have demonstrated that retroviral-mediated gene
correction could reconstitute the immune system in a murine XSCID model
expressing truncated c and that the transduced c enabled
cytokine-signaling in lymphoid cells by c-dependent receptors. These
findings have important implications because they suggest that
corrective gene transfer could be successful in XSCID patients who
carry mutations of the c gene, leading to the expression of
truncated c proteins. Of note, in the current series of experiments, we have used the identical vector and transduction conditions we had
previously used in c-null XSCID mice with similar overall efficacy,19 thus indicating that the presence of the
truncated mc did not hinder the therapeutic effects of gene
transfer. One possible explanation for these findings is that the
levels of wild-type c expression obtained in gene-corrected cells
were high enough to form sufficient numbers of functional receptors that could successfully compete with mutant c-containing receptors for cytokine binding.
Several patients with XSCID have been found to carry mutations leading
to truncated c protein expressed on the cell surface24; one patient treated with gene therapy expressed one of these mutant forms, which, however, did not seem to interfere with the successful outcome of the procedure. Although it is unclear whether these results
can be translated to all mutations compatible with residual surface
expression of c, the clinical experience with this particular patient complements our results in the c+-XSCID
animals and suggests that residual expression of c may not pose a
significant problem for gene therapy of XSCID. Additional studies are
needed to determine whether the same conclusions are applicable to all
c mutations (deletions or missense mutations) that are compatible
with residual surface expression of the c protein but that interfere
with normal cytokine signaling.
| |
Acknowledgments |
We thank Mrs Stacie M. Anderson for excellent technical assistance
and Dr David M. Bodine for insightful discussions and advice.
| |
Footnotes |
Submitted September 11, 2000; accepted November 20, 2000.
Supported in part by Japanese Society for the Promotion of
Science Research Fellowships for Japanese Biomedical and
Behavioral Researchers at the National Institutes of Health.
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.
Reprints: Fabio Candotti, Clinical Gene Therapy Branch,
National Human Genome Research Institute, National Institutes of
Health, 10 Center Dr, Bldg 10, Rm 10C103, MSC 1851, Bethesda, MD
20892-1851; e-mail: fabio{at}nhgri.nih.gov.
| |
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Abstract
Introduction