© The American Society of Gene & Cell Therapy
original article
HIV-specific Immunity Derived From Chimeric
Antigen Receptor-engineered Stem Cells
Anjie Zhen1,2, Masakazu Kamata1,2, Valerie Rezek1,2, Jonathan Rick1,2, Bernard Levin1,2, Saro Kasparian1,2,
Irvin SY Chen1–3, Otto O Yang2,3, Jerome A Zack1–3 and Scott G Kitchen1,2
Division of Hematology/Oncology, Department of Medicine, David Geffen School of Medicine at UCLA, Los Angeles, California, USA; 2UCLA AIDS
Institute and the Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research, David Geffen School of Medicine at UCLA, Los Angeles,
California, USA; 3Department of Microbiology, Immunology, and Molecular Genetics, David Geffen School of Medicine at UCLA, Los Angeles, California, USA
1
The human immunodeficiency virus (HIV)-specific cytotoxic T lymphocyte (CTL) response is critical in controlling HIV infection. Since the immune response does not
eliminate HIV, it would be beneficial to develop ways to
enhance the HIV-specific CTL response to allow longterm viral suppression or clearance. Here, we report
the use of a protective chimeric antigen receptor (CAR)
in a hematopoietic stem/progenitor cell (HSPC)-based
approach to engineer HIV immunity. We determined
that CAR-modified HSPCs differentiate into functional T
cells as well as natural killer (NK) cells in vivo in humanized mice and these cells are resistant to HIV infection
and suppress HIV replication. These results strongly suggest that stem cell-based gene therapy with a CAR may
be feasible and effective in treating chronic HIV infection
and other morbidities.
Received 27 March 2015; accepted 25 May 2015; advance online
publication 30 June 2015. doi:10.1038/mt.2015.102
INTRODUCTION
Immune-based therapies have emerged as a potentially powerful
approach toward the treatment of a variety of human diseases,
particularly chronic illnesses such as cancer or HIV. The genetic
modification of T cells or other immune cells to target a malignancy or viral infection holds significant promise over current
therapeutic strategies. Namely, these therapies potentially boost
better long-term disease control, lower toxicities, lower long-term
cost, and greater clinical efficacy. Recently, the use of chimeric
antigen receptors (CARs) to redirect T cells toward malignancies has become a high-profile method of treatment1 and represents a broad-based approach of engineered immunity that can
be used in a wide range of individuals, independent of transplantation antigen restriction. CAR-based approaches have involved
the redirection of peripheral T cells, particularly CD8+ T cells, to
target and kill cells expressing a tumor antigen.2,3 There are important limitations associated with the ex vivo genetic manipulation
of peripheral human cells that include the development of premorbid, dysfunctional cells that lack the ability to mount a sustained response following the extensive modification procedure
and engraftment.3–5 One prototype chimeric antigen receptor for
treating HIV infection is the CD4ζ chimeric antigen receptor. The
CD4ζ CAR molecule is a hybrid molecule consisting of the extracellular and transmembrane domains of the human CD4 molecule fused to the signaling domain of the CD3 complex ζ-chain.4–8
Thus when CD4 recognizes and engages HIV gp120 envelope protein on virally infected cells, the CAR-modified cell is triggered
and activated via ζ-chain signaling. CD4ζ CAR-modified T cells
were reported to inhibit viral replication and kill HIV-infected
cells in vitro.2,9 Clinical trials with CD4ζ CAR-modified T cells
showed that it is safe and that the transduced cells have prolonged
survival in vivo.3,7,8 However, the clinical efficacy of this approach
was hampered by lack of functional HIV responses in vivo following the modification of peripheral cells due to extensive and
damaging cell handling and genetic modification procedures. In
addition, expression of CD4 on gene modified T cells also rendered them susceptible to HIV-1 infection and elimination. Thus,
an approach that provides sustained production of functional
antigen-specific cells that are protected from infection could be
of significant benefit in the development of this type of therapy.
The use of human hematopoietic stem/progenitor cells
(HSPCs) instead of manipulated peripheral immune cells would
bypass many of these issues and provide long-term maintenance
of antigen-specific cells of multiple hematopoietic lineages. We
and others have previously demonstrated that HSPCs can be engineered with molecularly cloned T-cell receptors (TCRs) and can
further undergo development into functional, mature T cells following thymopoiesis.4,5,10–13 These modifications were assayed in
vivo using a humanized mouse model and resulted in a decrease
of HIV viral loads13 and reduced MART1 tumor size.11,12 However,
TCRs are restricted to individual human leukocyte antigens
(HLAs)(or “transplant antigens”), limiting their utility. The use of
a CAR would expand the breadth of this therapeutic approach by
bypassing the HLA restriction of cloned TCRs and overcoming
the virus ability to escape CTL responses, thus the CAR approach
could be employed in essentially any individual. However, it is
largely unknown if the expression of chimeric antigen receptor
would allow differentiation of multiple hematopoietic lineages.
Early studies done in mice using retroviral transduction of mouse
progenitor cells suggest that CD4ζ CAR expression may have
adverse effects on T-cell development.14,15 To date, only one study
tested the feasibility of modifying human HSPCs with an antiCD19 CAR16 and it remains unknown how CAR affects human
Correspondence: Scott G Kitchen, Division of Hematology/Oncology, Department of Medicine, David Geffen School of Medicine at UCLA, Los Angeles,
California, USA. E-mail: [email protected]
Molecular Therapy
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© The American Society of Gene & Cell Therapy
Engineered Anti-HIV Immunity From Stem Cells
hematopoietic differentiation and thymopoiesis and if CAR bearing cells generated in this fashion are functional in vivo.
In the present study, we explored the ability of CD4ζ CAR
to allow genetically modified HSPCs to produce multilineage
immune cells that target HIV infection in vivo using the humanized bone marrow-thymus-liver (BLT) mouse model. The BLT
humanized mouse model has the capability of generating the
broadest and most functional immune system of all current
humanized mouse models; and, cellular immune responses that
are generated to HIV infection closely mirror those in human.17–19
This allows us to assess the development and functionality of
the CD4ζ CAR-modified cells in vivo during HIV-1 infection.
Additionally, to protect engineered cells from HIV infection, we
combined CD4ζ CAR with efficient anti-HIV reagents20,21 to confer protection from HIV infection. Herein, we demonstrate the
feasibility and efficacy of utilizing a HSPC-based approach to
develop universally protective, multilineage HIV-specific cells in
vivo targeted toward the eradication HIV infection.
RESULTS
Construction and characterization of a protective
HIV-specific CAR
We constructed a lentiviral vector containing the CD4ζ CAR
(Figure 1a) as well as two antiviral genes, a small hairpin (sh)
RNA molecule specific to human CCR520 and a shRNA targeting specific HIV long terminal repeat (LTR) sequences (termed
sh516)21 (Figure 1b). The rationale for this is that CCR5 shRNA
would downregulate HIV coreceptor expression and the sh516
would target HIV RNA for degradation inhibiting productive
infection of the vector expressing cell. These additional measures
would likely protect the cells that express the CD4ζ CAR from
productive infection with HIV.20–23
We assessed the transduction and expression of the CD4ζ
CAR containing vector in sorted CD8+ T cells isolated from fresh
peripheral blood mononuclear cells. We compared cells transduced with the CCR5shRNA/sh516/CD4ζ CAR (Triple CAR)
containing vector to cells transduced with a vector containing only
the CD4ζ CAR or an empty vector (all expressing enhanced green
fluorescent protein (eGFP)) (Figure 2). Transduction and expression of the vector(s) resulted in extracellular CD4 expression and
we observed CCR5 knockdown in cells expressing the Triple CAR
a
vector (Figure 2a,b). As expected with CD4 expression on CD8+
T cells,24 we found that the expression of the CD4ζ CAR on CD8+
cells rendered them more susceptible to HIV infection compared
to a control vector lacking the CD4ζ CAR (Figure 2c). The presence of the antiviral shRNA in the triple CAR protected them from
infection (Figure 2c). In addition, we found that coincubation of
these cells with HIV-infected T1 cells resulted in the induction of
IL-2 and interferon-γ (IFN-γ) compared to cells coincubated with
uninfected T1 cells, indicating that the CD4ζ CAR is functionally
capable of inducing antiviral responses when expressed on CD8+
T cells (Figure 2d). Thus, we successfully developed a new vector that protects transduced cells from HIV infection as well as
expresses the CD4ζ CAR.
Hematopoietic development of triple CAR-modified
HSPCs in vivo
To determine if a CAR can be utilized in a stem cell-based
approach to produce immune cells capable of recognizing HIV,
we utilized a humanized mouse model in which multilineage
human hematopoiesis is recapitulated in an immunodeficient
mouse.17 In this system, mature, fully functional immune cells
are capable of developing from genetically manipulated human
HSPCs.11,13 Human HSPCs were transduced with the triple CAR
vector and were transplanted into immunodeficient nonobese
diabetic, severe combined immunodeficient, common γ-chain
knockout (γc−/−) (NSG) mice containing human fetal liver and
thymus tissue (Figure 3a). Following development of the transplanted tissue and genetically modified cells, we initially assessed
vector-expressing cells in different organs. We found that the
triple CAR was expressed on a significant number of cells in the
blood, spleen, thymus, and bone marrow of animals receiving vector modified HSPC’s, indicating that these cells undergo hematopoiesis in the transplanted animals (Figure 3b). Additionally,
we observed knockdown of CCR5 expression in vector-expressing cells in these animals, indicating that the shRNA specific to
CCR5 is functioning in vivo. We observed expression of the triple
CAR vector on T cells (CD45+CD19-CD5+), natural killer (NK)
cells (CD3-CD56+CD337+TCRαβ-), B cells (CD3-CD19+), and
myeloid cells (CD45+CD14+) in transplanted animals, indicating
that the genetically modified HSPCs are capable of multilineage
hematopoiesis in vivo (Figure 3c).
b
D
1
D
2
CD4
D
3
5′ LTR
H1
CCR5
sh1005
7SK
sh516
UbC
EGFP
2A
CD4-zeta
∆LTR
D
4
CD4 TM
CD3 Zeta
TCR
Figure 1 CD4 ζ chimeric antigen receptor. (a) Schematic presentation of CD4 ζ chimeric antigen receptor detailing the extracellular CD4 domains
D1-D4, the CD4 transmembrane portion (CD4 TM), and the T-cell receptor (TCR) CD3ζ signaling domain. (b) Schematic presentation of the triple
chimeric antigen receptor (CAR) vector showing the CCR5-specific shRNA (termed CCR5 sh1005), the HIV long terminal repeat-specific shRNA
(termed sh516), and the enhanced green fluorescent protein, the 2A peptide sequence, and the CD4ζ CAR genes.
2
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© The American Society of Gene & Cell Therapy
Engineered Anti-HIV Immunity From Stem Cells
a
CD4 ζ
EGFP
Q2
99.0
Q4
0
Q4
0
Q3
0.16
Q3
0
CD4
b
Q2
3.68
Q4
65.5
Q3
14.3
EGFP
Control
Q2
0.69
Q4
65.0
Q3
11.7
EGFP
CD4ζCAR+
Triple CD4ζCAR+
4.07
1.07
103
103
103
102
102
102
101
101
101
0
0
10
Q1
22.7
EGFP
0.31
CD8
Triple CD4 ζ
Q1
16.6
CCR5
Q3
25.8
10
Q3
0.55
CD4
CCR5
Q4
46.3
CCR5
Q2
8.51
0
Q4
0
CD4 ζ
EGFP
1
Q2
97.8
CD4
Q1
19.4
c
Q1
1.67
CD8
Q1
0.87
CD8
Q2
2.01
CD8
Q1
98.0
Triple CD4 ζ
2
10
3
0
0
1
10
2
10
3
10
101
0
102
103
Gag
d
Coincubated with
uninfected T1
IL-2
105
2.41
Coincubated with
HIV uninfected T1
0.52
105
104
104
103
103
102
0
102
0
92.8
4.28
0 102
103
104
105
12.6
17.7
49.2
0 102
20.5
103
104
105
IFNγ
Figure 2 In vitro validation assays of the protective TRIPLE CD4ζ chimeric antigen receptor (CAR) vector. (a) CD8 cells were purified from healthy
donors and transduced with either a GFP-only control vector, or CD4ζ CAR (no shRNAs), or the protective TRIPLE CAR vector (cells shown were gated
on eGFP expression). (b) CCR5 expression is down regulated by the protective CD4ζ CAR compared to the GFP-only or CD4ζ CAR (no shRNAs) controls. (c) HIV infection rates (gag+%) comparing HIV-1-exposed control CD8 cells to CD8 cells transduced with CD4ζ CAR (no shRNA) vector and the
TRIPLE CAR vector. Summary of fold decrease in gag+% cells comparing CD4ζ CAR CD8 cells and triple CAR CD8 cells from three healthy donors is
shown. (d) Cytokine production of the TRIPLE CAR vector transduced CD8 cells after coincubation with uninfected or HIV-infected T1 cells.
Expression of the CD4 ζ CAR suppresses endogenous
T-cell receptor recombination
Interestingly, while many of cells that express the triple CAR vector
that develop in vivo are T cells (47% ± 11.28%, see Supplementary
Figure S1), as determined by expression of CD5, CD7, or CD2,
there is a significant population of cells expressing these markers
that lack cell surface CD3ε. Further phenotypic analysis of this population indicates that these cells have lower levels of endogenous
Molecular Therapy
TCRαβ receptor expression (Figure 4a), which is necessary for
cell surface expression of CD3ε.25 Cell surface expression of CD3ε
is dependent on functional TCR expression in developing T cells
and we were interested if the CD4ζ CAR substituted for this TCR
during development and shut off the expression of the endogenous
TCR. We more closely examined these cells from animals transplanted with human HSPC modified with the triple CAR containing vector or a vector containing a deletion of the triple CAR solely
3
© The American Society of Gene & Cell Therapy
Engineered Anti-HIV Immunity From Stem Cells
a
1. Implant fetal thymus and liver
tissue
liv
thy
Irradiate
3 weeks
NSG
6–12
weeks
NSG
5. Analyze
human cell
reconstitution
4. i.v. Inject
Infect with
HIV-1
2. Sort CD34+ Stem Cells
6. Analyze
effects of
infection
3. Genetically modify stem cells
b
Human lymphocyte
1,200
CD4
Spleen
Blood
70.4
900
600
10
102
102
1
101
0
0
10
1
102
103
1.12
101
0
102
32.1
103
3.07
0.12
10
CCR5
10
102
103
Bone marrow
84.2
104
0.86
101
0
Thymus
105
4.44
3
0
22.7
0
62.6
103
10
96.7
CD45+
300
5.81
1.7
3
12.2
17.6
3
10
102
102
1
101
3
10
0
0
82.8
0 102
103
10
4
0
13.9
14
0.12
101
0
105
102
103
68.5
1.67
101
0
102
103
GFP
GFP
c
29.2
102
101
101
0
0
0
101
102
CD3
103
5.53
25.7
101
100
0
B cells
101
10
NK cells
1
0 10
2
10
10
CD19
3
73
0
6.64
1
10
2
10
10
CD337
3
10
1
102
CD8
0
0.35
101
0
0
0 10
MQs
0
32.7
36
102
103
CD3
TCRab
102
CD14
CD3
102
0
3.67
20
10
102
0
50.2
3
3
6/8.8
3
101
0 100
10
10
T cells
CD4
102
CD5
CD4
10
31.2
16.9
3
3
10
103
2.43
3
102
101
0
17
80.6
0
10
1
2
10
CD56
10
3
Figure 3 Construction and multilineage reconstitution of protective CD4ζ CAR hu-BLT mice. (a) Schematic illustrating the construction of triple chimeric antigen receptor (CAR) hu-BLT mice: CD34+ cells were purified from liver and transduced with lentiviruses containing the protective CD4ζ CAR and then transplanted into NSG mice with fetal liver stromal element and fetal thymus in matrigel. Three weeks
after transplantation, the transplant mice were sublethally irradiated (3Gy), previously frozen CD34+ cells were thawed and transduced and injected
into the mice where the cells engrafted in the bone marrow. Six to twelve weeks later, peripheral blood was collected and analyzed for human cell
reconstitution and the mice were infected with HIV-1. (b) Reconstitution of triple CAR hu-BLT mice expressing CD4ζ CAR. Cells were isolated from
the indicated organs and analyzed for their expression of human CD45 (leukocyte common antigen), vector expressing GFP, CD4, and CCR5 by
flow cytometry. (c) Multilineage hematopoietic reconstitution of triple CAR hu-BLT mice. Spenocytes from the triple CAR hu-BLT mice were analyzed
by flow cytometry and gated on human CD45+ and CD4+GFP+ triple CAR expressing cells. These cells were assessed for surface marker expression
identifying T cells (CD5+), B cells (CD3−CD19+), macrophages/monocytes (CD14+), and NK cells (CD56+TCRab−CD337).
expressing the eGFP marker protein. We observed a decrease in cell
surface CD3ε expression in cells expressing the CD4ζ CAR/eGFP
compared to cells expressing the eGFP control vector in the thymus
4
of transplanted animals (Figure 4b). When these thymocytes were
sorted and examined for the levels of T-cell receptor excision circles (TRECs), which indicate endogenous TCR rearrangement,
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Engineered Anti-HIV Immunity From Stem Cells
a
CD7+CD3+
CD7+CD3−
32.6
3
16
102
102
102
0
102
10
101
33.9
3
0
9.01
101
0
102
2.48
103
1
0
102
10
GFP103
GFP
control
62
103
102
101
101
0.97
CD5
0
0
0.74
1
2
10
10
40.8
TRIPLECD4CAR
10
3
0
5.94
101
58.3
102
102
1
101
10
0.73
0
0
0.23
2
10
10
103
67.3
0.8
0.76
101 102
68
103
1
102
TCR αβ
31.1
0
103
0
36.5
103
GFP+
36.3
102
0
0
4.97
TCR αβ
b
12
101
CD3
GFP
+
3
10
0
1
3
10
101
10
45.5
10
101
0
79.7
CD5
102
CD7
CD4
10
CD7+CD3
12.9
CD5
3
CD7+CD3−
24.4
29.9
1.55
3
10
0
103
0.58
1
10
102 103
CD3
d
102
101
0
***
20
0
103
gh
hi
FP
GFP
G
AR
C
C
102
le
1
Tr
ip
10
lo
w
0
0 10
le
-C
LE
IP
TR
40
Tr
ip
−
4C
D
4C
D
-C
LE
IP
FP
AR
G
AR
L
TR
C
TR
G
FP
FP
G
FP
G
L
TR
C
+
−
0.0
60
FP
0.5
CD3+% among
CAR+CD5+ cells
CD4
1.0
80
GFPlow GFPhigh
103
G
* P < 0.05
N.S.
AR
1.5
+
Fold change in TREC circle
c
Figure 4 Expression of triple chimeric antigen receptor (CAR) downregulates endogenous T-cell receptor (TCR) expression. (a) Splenocytes
from the CD4 ζ mice were assessed by flow cytometry and gated on triple CAR (CD4+GFP+) expressing cells. Expression of CD3, CD5, CD7, and TCR
αβ were assessed and analyzed by flow cytometry. (b) Thymocytes from the triple CAR-modified mice and control GFP mice were assessed for their
expression of T-cell markers CD5 and CD3 by flow cytometry. (c) Thymocytes from triple CAR-modified mice and control mice were sorted based
on CD5 and GFP expression. DNA was purified from the sorted cells and TCR rearrangement excision circle (TREC) was measured by real-time PCR.
(d) Levels of CD3 expression on triple CAR expressing cells (right panel) was analyzed separately by high (GFPhigh) or low GFP (GFPlow) expression, as
indicated by the respective gates (left panel).
we observed a significant decrease in TREC levels, of approximately 50%, in triple CAR vector-expressing cells compared to
cells expressing the control vector (Figure 4c). This indicates that
endogenous T-cell receptor rearrangement is at least partially shut
down when the CD4ζ CAR is expressed. These results are similar
to the results observed when a molecularly cloned, human MARTspecific T-cell receptor is introduced to human HSPCs.12 This suggests that the CD4ζ CAR is mimicking a normal T-cell receptor on
these newly produced cells. We also observed a reduction of CD3
expression on those cells expressing the greatest levels of the vector,
suggesting that higher levels of the CD4ζ CAR on the surface of
these developing cells more effectively turns off endogenous TCR
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rearrangement, presumably by providing a higher level of TCR ζ
chain signal to the developing cell (Figure 4d). In summary, these
data indicate that genetic modification of human HSPCs with a
triple CAR vector can result in multilineage hematopoiesis and
the production of HIV-specific T cells; a significant population of
which that have their endogenous T-cell receptor down regulated
and solely express the CD4ζ CAR molecule.
Triple CAR-modified cells differentiate into effector
cells in response to HIV infection in vivo
In order to assess the functionality of the new cells expressing
triple CAR, humanized mice transplanted with triple CAR cells
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© The American Society of Gene & Cell Therapy
Engineered Anti-HIV Immunity From Stem Cells
were then infected with HIV for 5 weeks. After this infection
time, virologic parameters and immune responses were assessed.
HIV infection resulted in the appearance of cells expressing triple CAR that possessed an effector phenotype (CD4+
eGFP+CD27−CD45RA+/−) that is not represented in the cells not
expressing the CAR (Figure 5a,b). This was similar to the types
a
b
CD45+GFP+
CD45+
3.19
65.3
44.9
30.8
80
103
% of EM&EMRA cells
103
CD45RA
of responses that we have observed in studies examining HIVspecific T-cell responses utilizing a molecularly cloned TCR
against HIV.13 In addition, these triple CAR expressing cells have
greater levels of the CD38 activation molecule (Figure 5c,d),
indicating the functional recruitment of these cells in antigenspecific T-cell responses to HIV. Cells were then assessed for
Naive
102
102
EMRA
101
101
CM
EM
4.88
0
26.6
2
0
10
10
0
15.2
3
9.11
2
0
10
10
**
60
40
20
0
3
CD45+
CD27
c
CD45+
50
103
102
102
101
101
0
0
14.9
10.9
101
0
102
103
35.1
*
30
20
10
8.95
0
5.99
101
e
10
2
103
0
CD45+
CD38
Uninfected T1 target cells
1.99
3
102
TNFα
10
CD4
40
CD38 MFI
CD4
17.6
3
10
d
CD45+GFP+
56.7
GFP+
101
0
0 10
10
1
10
2
10
CD45+GFP+
HIV infected T1 target cells
0.23
2.83
103
10
102
102
101
0
101
0
0.22
3
97.5
3.29
0
CD45+GFP+
0
3
0.31
101
2
10
88.1
103
0
8.81
101
102
103
IFNγ
GFP
f
g
7.32
10
103
8
GFPhigh
6
4
2
2
10
101
1
2
0
0
Uninfected T1
Infected T1
IFNγ MFI
TNFα+%
8
IFNγ+%
*
3
**
CD4CAR+
63.2
GFP
10
h
Uninfected T1
Infected T1
100
0
27.2
2.26
1
10
IFNγ
4
2
GFPlow
0
6
102
103
0
GFPhigh
GFPlow
Figure 5 Triple chimeric antigen receptor (CAR)-modified cells develop into effector phenotype and are activated after HIV infection. (a)
Splenocytes from HIV-1 infected triple CAR mice were assessed for naive (CD45RA+CD62L+), effector memory (EM) (CD45RA−CD62L−), central
memory (CM) (CD45RA−CD62L+), and effector memory RA (EMRA) (CD45RA+CD62L−) development. (b) Summary of % the EM and CM ratio among
CD45+ and CD45+GFP+ triple CAR cells from infected triple CAR mice. (c) Splenocytes from HIV-1-infected triple CAR mice were assessed for expression of activation marker CD38+. (d) Summary of CD38 mean fluorescence intensity (MFI) comparing CD45+ and CD45+GFP+ triple CAR cells from
infected triple CAR mice. (e) Splenocytes from HIV-1-infected triple CAR mice were accessed for their response to infected T1 target cells. Intracellular
production of IFNγ and TNFα from triple CAR were measured. (f) Summary of MFI of IFNγ from triple CAR cells cocultured with uninfected or infected
T1 target cells. (g) Summary of the TNFα MFI from triple CAR cells cocultured with uninfected or infected T1 target cells. (h) IFNγ production from
triple CAR cells coincubated with infected T1 target cells based on the level of GFP expression.
6
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© The American Society of Gene & Cell Therapy
virus-specific activation of antiviral responses during HIV infection. Splenocytes were removed from infected animals and were
then cocultured with a virally infected cell line or with uninfected cells. Shortly following exposure, cells expressing triple
CAR produced IFN-γ and tumor necrosis factor (TNF)-α in
response to HIV-infected cells and did not respond to uninfected
cells (Figure 5e–g). Interestingly, we found cells that express
high levels of eGFP and CD4ζ CAR expression responded more
robustly to infected cells in vitro and produced higher level
of IFN-γ than cells with lower level of CD4ζ CAR expression
(Figure 5h), despite their lack of endogenous TCR and CD3
expression (Figure 4d). These results indicate that cells carrying
the triple CAR were primed in vivo to elicit HIV-specific T-cell
responses following antigen encounter.
Triple CAR-modified cells are protected from HIV
infection and suppress HIV replication in vivo
Triple CAR expressing cells were then examined for evidence
of HIV infection in vivo by intracellular staining for HIV p24
Gag antigen. Significantly reduced levels of p24 Gag expression
were observed in cells expressing the triple CAR than in cells not
expressing the CAR (Supplementary Figure S2). This indicates
that these cells are protected from infection through the expression of the antiviral genes in the vector, allowing them to persist and respond against HIV. Interestingly, we found that some
mice had significant expansion of triple CAR cells (high expansion, >2.5-fold expansion of triple CAR expressing cells following infection) after HIV challenge (Figure 6a,b), while some did
not (low expansion, <2.5-fold expansion of triple CAR expressing cells following infection). We then assessed HIV serum viral
load (Figure 6c) as well as HIV DNA burden in peripheral blood
mononuclear cells (Figure 6d). We found that those mice that
had a greater expansion of cells expressing the triple CAR vector
had almost full suppression of HIV, while those animals whose
cells had lower levels of expansion did not have significant suppression of the virus (Figure 6c,d). This suggests that there is a
minimal response threshold by genetically modified, virus-specific cells necessary to suppress HIV replication. In addition to
lower viral loads, a better preservation of CD4/CD8 T-cell ratios
was observed in animals that had greater levels of cellular expansion of peripheral blood mononuclear cells expressing the triple
CAR vector (Figure 6e). The levels of cellular expansion correlated with suppression of the virus (Figure 6f). We found that triple CAR-modified mice that had poor responses to HIV infection
had correspondingly lower level of CD14+ antigen-presenting
cells in the blood (<2%) (Figure 6g), which may be due to suboptimal development of the myeloid lineage in the humanized
BLT mice model.19 On the contrary, mice that had successful suppression of HIV and greater expansion of triple CAR expressing
cells also had a higher percentage of CD14+ antigen-presenting
cells in the blood (5–9%). This is similar to the percentages of
CD14+ cells in normal human peripheral blood (2–10%).26 Thus,
the genetic modification of HSPCs with a lentiviral vector containing the CD4ζ CAR and protective antiviral genes can result
in the multilineage reconstitution of HIV-specific cells that are
protected from infection and can lower viral loads in vivo following virus challenge.
Molecular Therapy
Engineered Anti-HIV Immunity From Stem Cells
DISCUSSION
We have demonstrated that the modification of human HSPCs
with an HIV-specific CD4ζ CAR can allow the differentiation of
HIV specific T cells and cells of other lineages capable of lowering viral loads in vivo. The current study demonstrates that the
modification of HSPCs with a CAR, particularly one that contains
a molecule such as CD4 that is directly involved in the hematopoietic differentiation and selection process, allows the functional development of these cells. This has implications in the use
of other CAR molecules in HSPC-based approaches toward the
treatment of a variety of diseases.
Although redirecting anti-HIV immunity using a molecularly cloned TCR is promising, its application is restricted
by HLA type and many identified highly effective HIV CTL
utilize uncommon HLA alleles.27 In addition, recent research
by Deng et al.28 demonstrated that unless antiretroviral therapy is initiated early, the vast majority of latent viruses carry
CTL escape mutations that render these infected cells insensitive to naturally derived CTLs directed at common epitopes.
Engineering more effective CTL responses through the use of
a HSPC-based CD4ζ CAR therapy bypasses many of the problems associated with viral immune escape and HLA restriction by providing a broad-based and effective surveillance and
suppression of virally-expressing and latently-reactivated cells.
The HSPC-based therapy would also allow for long-lived and
renewable immunity that is capable of continuously generating anti-HIV cells which offers several advantages over current modalities, including those involving the redirection of
peripheral blood T cells that are more commonly being utilized
to treat a variety of malignancies.1 First, as it involves longlived stem cells, the approach described herein should require
only a single or limited number of administrations. The risk of
undesirable T-cell reactivity would be minimized, as stem cellderived T-cells will pass through thymic selection. As both CD4
and CD8 cells arise from CAR-transduced stem cells, there will
be both anti-HIV CD4- (helper) and CD8- (CTL) T-cell function. Finally, as a relatively high number of new, naive HIVspecific cells will be constantly renewed from stem cells, HIV
production from activated and infected reservoir cells would be
more effectively contained and prevented from systemic spread,
in part due to a potentially less “exhausted” phenotype of these
naive cells.
While we demonstrate the feasibility in the use of CARs in
a HSPC-based approach to target HIV disease in the humanized mouse, the overall approach utilizing autologous, genetically modified HSPCs in humans has been demonstrated to be,
thus far, a safe strategy.29 Based on this safety success, there are
multiple HSPC-based gene therapy clinical trails currently ongoing, primarily aimed at protecting cells from HIV infection
(ClinicalTrials.gov Identifiers: NCT01961063, NCT00569985,
NCT01177059, NCT01734850). Even though potential toxicities
related to the use of the CD4ζ CAR, including autoreactivity, cellular transformation, cytokine storms, or any other gross cellular
dysfunction or illness-inducing events, were not observed in our
studies, future studies directed toward the preclinical development of this approach should be focused on ascertaining these
potentially adverse events.
7
© The American Society of Gene & Cell Therapy
Engineered Anti-HIV Immunity From Stem Cells
hCD45+
105
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No. per 1,000 β globin
NS
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20
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CD4/CD8 ratio among T cells
c
2
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4
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10
15
HIV DNA copy per 1,000 β globin
20
Low
expansion
High
expansion
Figure 6 Triple chimeric antigen receptor (CAR) cells can successfully suppress HIV replication in vivo. (a) Representative figure of triple CAR
cells expansion upon HIV infection. (b) Fold expansion of GFP+CD4+ triple CAR expressing cells in peripheral blood 5 weeks after infection as assessed
by flow cytometry. (c) HIV viral load in serum 2 and 4 weeks postinfection. (d and e) Blood HIV DNA burden and CD4/CD8 ratio comparing control
and triple CAR mice that are infected with HIV-1. (f) Correlation of triple CAR expression cell expansion with viral burden in the peripheral blood.
(g) Percentage of human CD14+ cells among total human lymphocytes (CD45+) in the humanized mouse blood.
In the development and selection of CD4ζ CAR expressing
T cells that we observed, it is likely that the extracellular CD4
portion is interacting with HLA class II expressed by the thymic
stroma, which triggers positive selection of these cells. The observation that the cells that express the highest levels of the CD4ζ CAR
8
have the lowest level of CD3 and lower levels of TRECs suggests
these cells have the ability to ligate HLA II and send the strongest
signal to turn off endogenous TCR rearrangement (Figure 2c).
CD3 expression is dependent on the expression of a functional
TCR, and the signal generated through the CD4ζ CAR molecule
www.moleculartherapy.org
© The American Society of Gene & Cell Therapy
appears to turn off endogenous TCR rearrangement and expression. This may explain the lack of T-cell development observed in
previous mouse-based studies using CD4ζ CAR-modified progenitor cells as T cells were assessed by CD3 expression.14,15 Shut
down of endogenous TCR rearrangement could be functionally
beneficial in that a single TCR, the CD4ζ CAR, is on the surface of
approximately 50% of the cells and the theoretical cross-reactivity
of these cells toward another antigen is therefore reduced. In addition, CAR bearing T cells developed from HSPCs go through natural thymopoiesis, which eliminates self-reactive T cells and would
further limit the off-target adverse effects, and potentially the cytokine storms, observed in other CAR adoptive T-cell transfer therapies.30 Interestingly, the levels of the reduction in endogenous TCR
rearrangement observed with the CD4ζ CAR were similar to those
observed with a transgenic TCR to the MART1 antigen.12
The development of NK cells bearing the CAR is also of potentially important benefit in this type of stem cell-based approach
(see Figure 2c). NK cells, in addition to T cells, express the intracellular machinery to allow functional signaling through the TCR
ζ chain component of the CAR and have been previously shown to
direct CD4ζ CAR-modified mature NK cells to kill HIV-infected
target cells.31 The hematopoietic development of CD4ζ CARexpressing NK cells from genetically modified HSPCs can provide
a constant, innate immune response targeted to virus infection
capable of rapid cellular responses. These responses would further augment HIV immunity through the long-term production
of these HIV-specific cells and could contribute to NK-mediated
immunity and viral suppression at different anatomical sites.
The CD4ζ CAR has been found to be a safe reagent in multiple,
long-term clinical trials with over 500 patient years of clinical safety
data.7 Previous usage of CD4ζ CAR in adoptive transferred T-cell
therapy have had limited effects, potentially due to poor survival and
functionality of the transduced cells.32 Treatment was well-tolerated
and safe, but these studies were all confounded by the concurrent
administration of combination antiretroviral therapy; although, it
appeared in one trial that tissue viral replication was reduced.7 A significant problem was that the reinfused gene-modified T-cells were
premorbid and dysfunctional due to HIV infection in the individual
as well as massive ex vivo expansion. The net result was that they
persisted only at low levels following reinfusion. In addition, these
CD4ζ CAR expressing cells were susceptible to HIV infection due
to expression of the CD4ζ CAR itself in the absence of anything to
protect the cell from infection. Treatment through the redirection of
peripheral T cells in HIV-infected individuals inherently has a different set of issues than the, thus far promising, use of CARs in treating
malignancies, which are not confounded by the immune dysfunction created by HIV infection.1 It is highly likely that a approach such
as a HSPC-based strategy involving the CD4ζ CAR would be most
successful as a HIV cure strategy in combination with ART and/or
strategies that are designed to stimulate persistently infected cells,
allowing the newly produced CAR-containing cells to kill virally
expressing populations in different reservoirs.
Herein, we demonstrate the potential to generate HIV-specific
cells by redirecting T cells via stem cell gene therapy in a humanized mouse model. The triple CD4ζ CAR T cells undergo successful thymopoiesis and are protected from HIV infection. Most
importantly, these triple CD4ζ CAR expressing T cells could
Molecular Therapy
Engineered Anti-HIV Immunity From Stem Cells
differentiate from a naive phenotype into an effector phenotype,
produce multifunctional T-cell responses, expand upon infection,
and effectively suppress HIV replication in vivo. This suggests that
the effector function of the triple CD4ζ CAR T cells plays important role in suppressing HIV infection. For mice that had weak
expansion of CD4ζ CAR T cells, our data suggest that this is likely
due to lower reconstitution of antigen-presenting cells, which provide crucial costimulatory signals for T-cell activation, in these
mice. A current line of investigation is aimed at enhancing CARtriggered response through the enhancement of APC development/function in this system. In sum, our results demonstrate the
feasibility of modifying human HSPCs with protective CD4ζ CAR
as a therapeutic approach against HIV infection.
MATERIALS AND METHODS
Construction of triple CAR BLT mice. Triple CAR BLT mice were constructed similarly to previously reported HIV TCR-modified humanized
mice.13 In short, CD34+ cells were purified via magnetic activated cell sorting with CD34-specific beads (Miltenyi, Auburn, CA) from freshly obtained
fetal liver tissue. Cells were then transduced overnight with lentiviruses
containing the control vector or the protective triple CAR utilizing a retronectin-based procedure.13 Cells were then transplanted into NSG mice with
fetal liver stromal and fetal thymus in derived from the same tissue placed
in matrigel. Three weeks after transplantation, the transplant mice were sublethally irradiated (3Gy), previously frozen CD34+ cells were thawed and
transduced with the appropriate vector and 0.5 million transduced CD34+
cell were injected into each mouse. Transduction of the CD34+ cells was
assessed by culturing 50,000 vector-treated CD34+ cells in liquid culture for
3 weeks. We typically obtained ~30% transduction with the triple CAR or
control lentivirus vectors. Six to twelve weeks later, peripheral blood was
collected and analyzed for human cell reconstitution; and, following confirmation of reconstitution, the mice were then infected with HIV-1NL4-3
(300ng p24, as determined by enzyme-linked immunosorbent assay).
Human fetal tissue was purchased from Advanced Biosciences
Resources (Alameda, CA) or from Novogenix Laboratories (Los Angeles,
CA) and was obtained without identifying information and did not
require Institutional Review Board approval for its use. Animal research
described in this manuscript was performed under the written approval
of the UCLA Animal Research Committee (ARC) in accordance to all
federal, state, and local guidelines. Specifically, these studies were carried
out under strict accordance to the guidelines in The Guide for the Care
and Use of Laboratory Animals of the National Institutes of Health and
the accreditation and guidelines of the Association for the Assessment
and Accreditation of Laboratory Animal Care International under UCLA
ARC Protocol Number 2010-038-02B. All surgeries were performed
under ketamine/xylazine and isofluorane anesthesia and all efforts were
made to minimize animal pain and discomfort.
Antibodies and flow cytometry. The following antibodies were used in flow
cytometry: CD45, CD2, CD7, CD5, CD3, CD4, CD8, CD45RA, CD62L,
CD38, CD19, CD14, CD337, CD56, TCRαβ (eBiosciences, San Diego, CA),
and anti-HIV-1 core antigen clone KC57 (Beckman Coulter, Brea, CA). Cell
surface markers are conjugated to either FITC, PE, PerCP-Cy5.5, PE-Cy5,
PE-Cy7, EVD, APC, APC-eflou780, alexa700, eflour405, Pacific orange
or pacific blue in appropriate combination. The cells were acquired using
LSRFortessa flow cytometer (BD biosciences, San Jose, CA) and FACSDiva
software. Data were analyzed using FlowJo software (Ashland, OR).
Lentiviral vector production. The lentivirus-based GFP control vector and triple CD4ζ CAR vector were produced in 293FT cells using the
Invitrogen (Grand Island, NY) ViraPower Lentiviral Expression system
with pCMV.ΔR8.2.Δvpr packaging plasmid and the pCMV-VSV-G envelope protein plasmid as previously described.10
9
Engineered Anti-HIV Immunity From Stem Cells
Purification of viral RNA and reverse transcription in mouse plasma.
Mouse peripheral blood was drawn by retro-orbital bleeding into glass capillary tubes coated with 330 mmol/l ethylenediaminetetraacetic acid (EDTA)
(Gibco, Grand Island, NY), and 3% sterile human serum albumin (Baxter
Healthcare, Deerfield, IL). Plasma was obtained by centrifuging the blood
at 6,000 rpm for 3 minutes. Viral RNA was extracted from plasma with
the QIAAmp Viral RNA extraction Kit (Qiagen Venlo, The Netherlands).
Afterwards, cDNA were generated from viral RNA using high capacity
reverse transcription kit (Life Technologies, Grand Island, NY).
DNA extraction of peripheral blood. Mouse peripheral blood was drawn
by retro-orbital bleeding into glass capillary tubes coated with 330 mmol/l
EDTA (Gibco), and 3% sterile human serum albumin (Baxter Healthcare).
After red blood cell (RBC) lysis, DNA were extracted by phenol–chloroform extraction as previously described.33
Real-time polymerase chain reaction (PCR) of viral cDNA and DNA.
Real-time PCR was performed with TaqMan Real time PCR Master
Mix (Life Technologies) with the following primers and probe was
used for real-time PCR of viral cDNA and DNA. FL-2 forward primer:
5′-CAATGGCAGCAATTTCACCA-3′; FL-2 Rev primer: 5′-GAA
TGCCAAATTCCTGCTTGA-3′; Probe: 5′-(6-FAM)CCCACCAACAGG
CGGCCTTAACTG(Tamra-Q)-3′. These primers anneal in the pol region of
the genome (4,577–4,653), within the first intron. HIV standards were made
by linearizing pNL4.3 with EcoRI and quantitated by spectrophotometry.
Quantitation of TCR-rearrangment excision circles. Thymocytes from
triple CAR or GFP vector-modified mice were sorted on a FACSAria
(BD Biosciences) based on their expression of GFP and CD5. DNA was
then extracted from sorted cells using phenol/chloroform. Real-time PCR
was used to quantify TREC levels normalized to β-globin as described
previously.34
Cytokine assay. CD8+ cells transduced with the control vector or the triple CD4ζ CAR vector, or splenocytes from HIV-1-infected triple CARcontaining mice were coincubated with uninfected or HIV-1-infected T1
target cells overnight and treated with GolgiPlug (BD Biosciences) for an
additional 6 hours. Intracellular production of IFN-γ and TNF-α from
CD4ζ CAR expressing cells were measured by intracellular staining and flow
cytometry13
SUPPLEMENTARY MATERIAL
Figure S1. In vivo T cell differentiation of TRIPLE CD4ζ CAR cells in
humanized mice.
Figure S2. TRIPLE CD4ζ CAR cells are protected from in vivo HIV-1
infection.
ACKNOWLEDGMENTS
This work was funded by grants from the NIAID/NIH, grant no.
RO1AI078806, the UCLA Center for AIDS Research (CFAR), grant no.
P30AI28697, the California Institute for Regenerative Medicine, grant no.
TR4-06845, and the UC Multi-campus Research Program and Initiatives,
California Center BD Biosciences for Antiviral Drug discovery (CCADD)
and California HIV/AIDS research Program F12-LA-215 (to AZ).
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