Group Title: Retrovirology 2004, 1:37
Title: Alteration of T cell immunity by lentiviral transduction of human monocyte-derived dendritic cells
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Title: Alteration of T cell immunity by lentiviral transduction of human monocyte-derived dendritic cells
Series Title: Retrovirology 2004, 1:37
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Creator: Chen X
He J
Chang LJ
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Source Institution: University of Florida
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Retrovirology


B
BioMed Central


Research


Alteration of T cell immunity by lentiviral transduction of human
monocyte-derived dendritic cells
Xiaochuan Chen, Jin He and Lung-Ji Chang*


Address: Department of Molecular Genetics and Microbiology, Powell Gene Therapy Center, McKnight Brain Institute, University of Florida
College of Medicine Gainesville, FL 32610-0266, USA
Email: Xiaochuan Chen xchen@ufl.edu; Jin He jinhe@ufl.edu; Lung-Ji Chang* lchang@mgm.ufl.edu
* Corresponding author


Published: 01 November 2004
Retrovirology 2004, 1:37 doi:10. 1186/1742-4690-1-37


Received: 28 June 2004
Accepted: 01 November 2004


This article is available from: http://www.retrovirology.com/content/1/1/37
2004 Chen et al; licensee BioMed Central Ltd.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.



Abstract
Background: Dendritic cells (DCs) are professional antigen-presenting cells that play important
roles during human immunodeficiency virus type I (HIV-1) infection. HIV-1 derived lentiviral
vectors (LVs) transduce DCs at high efficiency but their effects on DC functions have not been
carefully studied. Modification of DCs using LVs may lead to important applications in
transplantation, treatment of cancer, autoimmune and infectious diseases.
Results: Using DCs prepared from multiple blood donors, we report that LV transduction of DCs
resulted in altered DC phenotypes and functions. Lentiviral transduction of DCs resulted in down-
regulation of cell surface molecules including CD I a, co-stimulatory molecules CD80, CD86, ICAM-
I, and DC-SIGN. DCs transduced with LVs displayed a diminished capacity to polarize naive T cells
to differentiate into Thl effectors. This impaired Thl response could be fully corrected by co-
transduction of DCs with LVs encoding interleukin-12 (IL-12), interferon-gamma (IFN-y), or small
interfering RNA (siRNA) targeting IL- 10.
Conclusions: DCs transduced with LVs in vitro displayed diminished Th I functions due to altered
DC phenotypes. Our study addresses an important issue concerning lentiviral infection and
modification of DC functions, and provides a rational approach using LVs for immunotherapy.


Background
During HIV-1 infection, an increase in DC-SIGN and
CD40 has been reported, as has a decrease in the expres-
sion of CD80 and CD86 in dendritic cells (DCs) of lym-
phoid tissue [1]. Although some suggest that HIV-1
infection reduces the production of IL-12 by DCs,[2] oth-
ers have shown that DCs derived from HIV-1-infected
individuals express both IL-12 and IL-10 at levels similar
to those in non-infected individuals[3] While these stud-
ies have explored the effects of wild-type HIV-1 on DC
functions, the possible effects of HIV-1-derived lentiviral


vectors (LVs) on DC functions have not been well charac-
terized [1].

LVs are useful gene transfer tools that can efficiently target
many types of cells including DCs. As important immune
modulating cells for immunotherapy and vaccine applica-
tions, DCs play critical roles in activating the host
immune response. DCs can capture, process, and present
foreign antigens, migrate to lymphoid-rich tissues, and
stimulate antigen-specific immune responses [4]. DCs
present a variety of signals to stimulate T cells and initiate
immune response; these signals involve multiple


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signaling mediators, including MHC molecules harboring
antigenic peptides (signal 1), the co-stimulatory mole-
cules CD80, CD86, and ICAM-1 (signal 2), and cytokines
such as IL-12, IL-4, and IL-10 (signal 3) [5].

Engagement between DCs and T cells not only stimulates
T-cell proliferation, but also polarizes differentiation of
naive T helper (Th) cells into IFN-y-producing Thl or IL-
4-producing Th2 effector cells [6,7]. Production of IL-12
by DCs early in an immune response is critical for polari-
zation of CD4+ T cells toward Thl function, which is
essential for the clearance ofintracellular pathogens. IL10,
on the other hand, suppresses IL-12 production from DCs
and diminishes the commitment of Th differentiation.
Besides cytokine signaling, there is accumulating evidence
that co-stimulatory molecules and adhesion molecules
such as CD80, CD86, and ICAM-1 not only engage in T-
cell stimulation, but also direct the differentiation of
naive T cells [8-10].

Efficient gene transfer into DCs without cytotoxicity has
always been difficult [11,12]. LVs transduce DCs at high
efficiencies with little to no cytotoxicity, and the trans-
duced DCs retain their immature phenotype, are able to
respond to maturation signals, and maintain immunos-
timulatory potential in both autologous and allogeneic
settings [13-16]. In this study, we carefully analyzed cellu-
lar response to LV transduction by evaluating changes in
DC phenotypes using monocyte-derived DCs prepared
from more than 40 blood donors. We investigated the
function of DCs to polarize naive T cells to Th effectors
after LV infection. Our results demonstrated altered DC
functions after LV gene transfer. Most importantly, we
illustrated effective modulation of DC immunity by LV
expression of different cytokines or siRNA molecules.

Materials and Methods
Generation of monocyte-derived dendritic cells
Peripheral blood mononuclear cells (PBMCs) from
healthy donors (Civitan Blood Center, Gainesville, FL)
were isolated from buffy coats by gradient density centrif-
ugation in Ficoll-Hypaque (Sigma-Aldrich, St. Louis, MO)
as previously described [17]. DCs were prepared accord-
ing to the method ofThumer et al. [18], with the follow-
ing modifications: On Day 0, five million PBMCs per well
were seeded into twelve-well culture plates with serum-
free AIM-V medium (Invitrogen Corp. Carlsbad, CA). The
PBMCs were incubated at 370C for 1 hr and the non-
adherent cells were gently washed off; the remaining
adherent monocytic cells were further cultured in AIM-V
medium until Day 1. The culture medium was removed
with care not to disturb the loosely adherent cells, and 1
ml per well of new AIM-V medium containing 560 u/ml
of recombinant human GM-CSF (Research Diagnostic
Inc., Flanders, NJ) and 25 ng/ml of IL-4 (R&D Systems,


Minneapolis, MN) was added and the cells were cultured
at 37C and 5% CO2. On Day 3, 1 ml of fresh AIM-V
medium containing 560 u/ml of GM-CSF and 25 ng/ml of
IL-4 was added to the culture. On Day 5, the non-adherent
cells were harvested by gentle pipetting. After washing, the
DCs were frozen for later use or used immediately.

Lentiviral vector construction and preparation
MLV and LVs were constructed as described previously
[19,20]. The self-inactivating pTYF vectors expressing
CD80, CD86, GM-CSF, and IL-12 genes under the EFla
promoter control were constructed by inserting cDNAs
that have been previously functionally characterized [21-
23]. The cDNA of ICAM-1 was derived from pGEM-T-
ICAM-1 kindly provided by Dr. Eric Long. The cDNAs of
Flt3L, CD40L, and IL-7 were amplified by RT-PCR using
the primers listed below with a modified eukaryotic trans-
lation initiation codon (CCACC-AUG): Flt3L sense 5'-TIT
CTA GAC CAC CAT GAC AGT GCT GGC GCC AG-3' and
antisense 5'-AAG GAT CCT CAG TGC TCC ACA AGC AG-
3'; CD40L sense 5 '-TIT CTA GAC CAC CAT GAT CGA AAC
ATA CAA C-3' and antisense 5'-TTG AAT TCT TAT GTT
CAG AGT TTG AGT AAG CC-3'; IL-7 sense 5'AAG CGG
CCG CCA CCA TGT TCC ATG TIT CTT-3' and antisense
5'-TTC TCG AGT TAT CAG TGT TCT TTA GTG CCC ATC-
3'.

The LVs were produced and concentrated as described pre-
viously [20]. Lentiviral siRNA vectors were generated as
previously described, using four oligonucleotides. IL-
10i#1: sense 5'-GAT CCC CAG CCA TGA GTG AGT TTG
ACT TCA AGA GAG TCA AAC TCA CTC ATG GCT TIT
TTG GAA A-3' and antisense 5'-AGC TIT TCC AAA AAA
GCC ATG AGT GAG TIT GAC TCT CTT GAA GTC AAA
CTC ACT CAT GGC TGG G-3'; ILl Oi#2: sense 5'-GAT CCC
CGG GTT ACC TGG GTT GCC AAT TCA AGA GAT TGG
CAA CCC AGG TAA CCC TIT TTG GAA A-3' and antisense
5'-AGC TIT TCC AAA AAG GGT TAC CTG GGT TGC CAA
TCT CTT GAA TTG GCA ACC CAG GTA ACC CGG G-3'
[24].

Lentiviral transduction of immature DCs and DC
maturation
We plated Day-5 immature DCs at 5 x 105 per well in a 24-
well plate containing 200 gil of medium supplemented
with GM-CSF (560 u/ml) and IL-4 (25 ng/ml). DC infec-
tion was carried out by adding concentrated LVs to the
cells at a multiplicity of infection (MOI) of 50-100
(_105-106 transducing units/ng of p24) as previously
described [25]. The infected cells were incubated at 37C
for 2 hr with gentle shaking every 30 min, then 1 ml of DC
medium was added and the culture was incubated with
the viral vectors for an additional 12 hr. DC maturation
was induced by adding lipopolysaccharide (LPS) at a final
concentration of 80 ng/ml and TNF-a at a final


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concentration of 20 u/ml and incubated for 24 hr. To col-
lect mature DCs, the cells were treated with AIM-V
medium containing 2 mM EDTA at 37 C for 20 min, and
washed three times with PBS.

Antibody staining and flow cytometry
For analysis of cell-surface marker expression by flow
cytometry, we incubated DCs for 10 min with normal
mouse serum and then 30 min with fluorochrome-conju-
gated anti-human monoclonal antibodies. In different
experiments, these antibodies included HLA-ABC (Tu149,
mouse IgG2a, FITC-labeled, Caltag Laboratories, Burlin-
game, CA); HLA-DR (TU36, mouse IgG2b, FITC-labeled,
Caltag Laboratories); CDla (HI49, mouse IgGlk, APC-
labeled, Becton Dickinson Pharmigen, San Diego, CA);
CD80 (L307.4, mouse IgGlk, Cychrome-labeled, Becton
Dickinson);CD86 (RMMP-2, Rat IgG2a, FITC-labeled,
Caltag Laboratories); ICAM-1 (15.2, FITC-labeled, Calbi-
ochem); DC-SIGN (eB-h209, rat IgG2a, APC-labeled, eBi-
oscience, San Diego, CA); CD1 Ic (Bly-6, mouse IgG1, PE-
labeled, BD Pharmigen); CD40 (5C3, mouse IgG1, Cy-
chrome-labeled, Becton Dickinson); CD123 (mouse
IgG1, PE-labeled, BD Pharmigen); and CD83 (HB15e,
mouse IgG1, R-PE-labeled, Becton Dickinson). We
included the corresponding isotype control antibody in
each staining condition. After two washes, the cells were
resuspended and fixed in 1% paraformaldehyde in PBS
and analyzed using a FACSCalibur flow cytometer and the
CELLQUEST program (Becton Dickinson). The live cells
were gated by forward- and side-light scatter characteris-
tics and the percentage of positive cells and the mean flu-
orescence intensity (MFI) of the population were
determined.

FACS sort of lacZ-positive cells
The lentiviral siRNA vector-transduced cells co-expressing
nuclear lacZ gene were separated from un-transduced cells
by staining with fluorescent LacZ substrate and sorted by
FACS. To label the lacZ-positive cells, we resuspended
cells in 100 ul medium and added 100 ul of FDG (fluores-
cein di-beta-D-galactopyranoside) working solution (2
mM) which was diluted from a 10 x stock FDG solution
(20 mM). The stock solution was made by dissolving 5 mg
FDG (MW 657, Molecular Probe, Eugene, OR) in a 1:1
mixture of DMSO/ethanol and mixing with ice-cold
ddHO2 to make an 8:1:1 ddHO2/DMSO/ethanol solu-
tion. The cells were incubated in 37C water bath for 1-
1.5 min, and diluted with 10-fold volume of cold medium
and kept on ice until FACS sorting.

Preparation of naive CD4+ T cells
The CD4+ T cells from PBMCs were collected by negative
selection, using a CD4+ T cell isolation Rosette cocktail
(StemCell Technologies, Vancouver, BC) according to the
manufacturer's instructions. Briefly, we centrifuged 45 ml


of buffy coat (approximately 5 x 108 PBMCs) in a sterile
200-ml centrifuge tube with 2.25 ml of the CD4+ T cell-
enrichment Rosette cocktail at 25 C for 25 min. Thereaf-
ter, 45 ml of PBS containing 2% FBS was added to dilute
the buffy coat. After gentle mixing, we layered 30 ml of the
diluted buffy coat on top of 15 ml of Ficoll Hypaque in a
50-ml centrifuge tube and centrifuged for 25 min at 1,200
g. Non-rosetting cells were harvested at the Ficoll interface
and washed twice with PBS (2% FBS), counted, and cryo-
preserved in aliquots in liquid nitrogen for future use. The
purity of the isolated CD4+ T cells was consistently above
95%. The CD4+CD45RA naive T cells were purified based
on negative selection of CD45RO- cells using the MACS
(Miltenyi Biotec, Auburn, CA) magnetic affinity column
according to the manufacturer's instructions.

In vitro induction of Th functions and intracellular
cytokine staining
The in vitro DC:T cell co-culture method was modified
based on Caron et al[26]. Briefly, we co-cultured purified
naive CD4 T cells with allogeneic mature DCs at different
ratios (20:1 to 10:1) in serum-free AIM-V media. On day
5, 50 u/ml of rhIL-2 was added and the culture was
expanded and replenished with fresh AIM-V medium con-
taining rhIL-2 every other day for up to 3 weeks. After day
12, we washed the quiescent Th cells and re-stimulated
them with PMA (10 ng/ml or 0.0162 jiM) and ionomycin
(1 jig/ml, Sigma-Aldrich) for 5 hr, adding Brefeldin A (1.5
jig/ml) during the last 2.5 hr of culture. We then fixed,
permeablized, and stained the cells with FITC-labeled
anti-IFN-y and PE-labeled anti-IL-4 mAb (Pharmigen, San
Diego, CA). The cells were analyzed in a FACSCalibur
flow cytometer (BD Biosciences, San Diego, CA).

DC-mediated mixed lymphocyte reaction
We co-cultured serial dilutions of DCs, from 10,000 cells
per well to 313 cells per well, with 1 x 105 allogeneic CD4
T cells in a 96-well U-bottomed plate in a total volume of
200 il for 5 days. The proliferation of T cells was moni-
tored by adding 20 il of the CellTiter96 solution to each
well according to the manufacturer's instruction
(Promega). The cells were further cultured for 4 hr before
reading the OD490value using a microplate reader (EL808,
BIO-TEK Instrument Inc.,Winooski, VT).

Results
LVs altered surface marker expression in peripheral blood
monocyte-derived DCs
To investigate the effects of lentiviral vectors (LVs) on
DCs, we transduced monocyte-derived DCs with LVs
encoding different reporter genes. The efficiency of LV
transduction of DCs is illustrated by a reporter gene assay
(Fig. 1A). The DCs were derived from healthy donors'
PBMCs, and on day 5 (d5) of culture, the immature DCs



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PBMC


dO dl IL-4
GM-CSF


+LV +LPS, TNFa.

12d5(iDCs) d6 d(mDC)
dm I d7 I II mC
d5(imDCs) d6 d7(mDC)


D7 monocyte-derived dendritic cells

*" -.s" ra ^ '
i *--' we a


0

1-*'- ^'

fl eQ

Mock


LV-PLAP


Mock Empty LV- MLV
LV PLAP



CDllc 4

CD123 j Wi L

CDla

CD40 s I'W' k

ICAM-1i

CD62L

CDSO j t n

CD83

CD86

DC-SGN.7

HILA-ABC

HLA-DR


Mock Empty LV- MLV
LV Cre




Is3 -12,3 j-S .s .7

|Li2k27H-' tE^L



t^^^y^.^J -- : ^i '"^ *^u -




^ii^;E

'14"^ tt8:* WtA.; O'


Figure I
LV transduction of DCs and analysis of surface marker expression. PBMC-derived DCs were infected with LV on Day
5 (d5) and after maturation, co-cultured with naive CD4 T cells for 1-2 weeks before intracellular cytokine staining (ICCS) and
flow cytometry (FACS) analysis. The d5 DCs were transduced by LV-PLAP and 48 hr later, analyzed by PLAP enzyme assay. (B)
FACS analysis of DC surface markers after viral transduction. The d5 DCs were transduced with LV carrying PLAP or Cre as
reporter gene, MLV vectors, empty LV, or no vector controls (mock) and treated with TNF-a plus LPS. The cell surface mark-
ers were stained with antibodies and analyzed by FACS. The numbers represent mean fluorescence index (MFI) and the results
are representatives of six experiments.



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{


Retrovirology 2004, 1:37








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Table I: Surface marker profile of DCs transduced with LVs or MLV vectors.


Geometrical Mean Fluorescence SD


Surface Marker

CD IIc
CD 123
CDla
CD40
ICAM- I
CD62L
CD80 (B7-1)
CD83
CD86 (B7-2)
DC-SIGN
H LA-ABC
HLA-DR


Mock


48.8 3.2
13.0 0.4
27.3 I.I
8.6 0.1
462.6 57.5
3.3 0.1
9.9 0.9
5.8 0.3
39.6 3.5
62.7 4.5
13.9 1.3
31.5 0.8


Empty LV

47.2 1.3
13.4 0.8
27.6 2.9
8.9 0.6
376.5 30.1
3.2 0.03
10.6 0.7
5.8 0.1
39.6 2.5
55.7 0.4
15.8 1.0
28.6 2.2


LV

52.3 2.3
14.9 0.6
21.5 0.2*
8.6 0.1
179.5 3.4***
3.7 0.1
9.3 0.2*
6.4 0.01
31.4 0.4*
50.6 1.5*
14.6 0.3
26.9 0.4


MLV

55.3 1.1
15.7 0.1
31.0 0.3
9.0 0.3
498.5 6.9
3.3 0.4
1 1.3 0.4
6.0 0.3
47.3 1.5
68.6 4.1
17.2 0.9
33.2 1.7


Results are presented as geometrical mean fluorescence after FACS. Asterisks (*) denote significance of difference by Student t-test (*P < 0.05, **P
<0.0 1, ***P < 0.001 ).


(imDC) were infected with LV-PLAP (encoding placenta
alkaline phosphatase). Analysis of PLAP activity on day 7
demonstrated transduction efficiency of> 80% (Fig 1B).

DC functions through surface receptor signaling
To see if LVs affected DC surface marker expression, we
examined the expression profile of surface molecules on
DCs by antibody immunostaining. We transduced PBMC-
derived imDC with mock (control 293 supematants) vec-
tors, empty LV particles, LV, and MLV carrying a reporter
gene. After induction of maturation with LPS plus TNF-a,
we harvested the DCs for antibody staining and FACS. The
results are shown in Fig. 1C and summarized in Table 1.
Among the surface molecules tested, CD1a, CD80, CD86,
ICAM-1, and DC-SIGN were down-regulated after LV
transduction, but not after transduction with empty LV or
MLV. The same result was obtained using different prepa-
rations of LVs carrying either PLAP or Cre as the reporter
gene.

LV transduction impaired DC-mediated Th I immunity
It has been reported that retroviral infection induces up-
regulation of Th2 cytokines including IL-10 and impairs
DC maturation [27,28]. Because HIV causes immune sup-
pression and the preceding results showed that LV infec-
tion altered the surface marker expression profile of DCs,
we suspected that LV infection might also affect DC acti-
vation of T cells. To test this, we set up an in vitro immu-
nity assay using co-culture of human DCs and naive T
cells.

We generated DCs from PBMCs and infected the d5 DCs
with LV carrying a reporter gene. To characterize the func-
tion of DCs, we purified naive CD4+ T cells from healthy


donors' blood and co-cultured the T cells with allogeneic
monocyte-derived DCs treated with TNFa and LPS to
induce maturation, as illustrated in Fig. 2. The co-cultured
T cells were allowed to expand and rest for more than 7
days after DC priming. To analyze Th response, on days 7
and 9 we reactivated the resting T cells with ionomycin
and PMA, and subjected the T cells to ICCS using antibod-
ies against IFN-y and IL-4. We found that the IFN-y-pro-
ducing Thl cell populations were dramatically reduced
when incubated with DCs transduced with LVs, from 72%
(day 7) and 75% (day 9) for the control to 27% (day 7)
and 22% (day 9) for the LV-transduced DCs. The Th2 pop-
ulations remained essentially unchanged (Fig. 2). In naive
T cells the Thl response is regulated by the "master tran-
scription regulators" T-bet and GATA-3.[291 Analysis ofT-
bet and GATA-3 expression in T cells after coculture with
LV-transduced DCs showed decreased expression of both
T-bet and GATA-3 RNA, and the relative T-bet expression
correlated with the Th differentiation according to ICCS of
T cells after 8 days of co-culture (data not shown).

Up-regulation of CD80 and CD86 expression did not
restore DC functions
Because T cell co-stimulatory molecules are important
mediators of DC functions, the down-regulation of CD80
and CD86 in DCs after LV transduction might contribute
to the observed Thl impairment. To examine this possi-
bility, CD80 and CD86 were up-regulated in DCs using
LVs encoding these two genes to see if the impaired Thl
response could be corrected. The LVs encoding human
CD80 and CD86 were constructed as shown in Fig. 3A.
The functions of these CD80 and CD86 genes have been
previously demonstrated in an in vivo study [21]. DCs
were transduced with LVs expressing a reporter, CD80 or



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+LV
sys12hr
- 10 I --+


A ^PBMC

ep e) o l I


dl IL-4 d5
GM-CSF


+ LPS, TNFa



d6


+ naive CD4 T

Lsei2 --l Thl/Th2
d7 d14- d16


B DC/ coculture Day
DC/T coculture Day7


Day9


Mock

S ICCS020327.007






0 50 100 150 200 2'
0


LV

ICCSO:20327.009






0 5so 100 IS 200 250


Mock


LV
S ICCS020329.009






0 50 100 150 200 250


SSC
S ICC020327.007 r ICCS020327.00 9 ICCS020329.007 ICCS020329.009
0.6 3.6 0.5 1.6 1.4 5.7 0.8 1.9




1. 10 10 '" 10 101 1 10 10 1o 10 102 103 10 i4 101 i 02 I0 10




Figure 2
Impaired Th I response induced by LV-transduced DCs. We analyzed T helper function by using DC:T cell co-culture
and IL-4 and IFN-ylCCS. Immature DCs were infected with mock (293T supernatants) or LV on d5 and treated with LPS and
TNFa. The DCs were harvested and co-cultured with naive CD4+ T cells at a DC:T cell ratio of 1:10. On Day 7 amd 9 after
co-culture, the cells were re-stimulated and the T helper cell populations were examined by INF-y and IL-4 antibody ICCS as
described in the Materials and Methods. The percentages of cell populations are indicated in the FACS quadrants. The results
are representative of four independent experiments.


CD86 gene, and then treated with LPS and TNF-a 12 hr
later. Thirty-six hours after LV transduction, we analyzed
the transduced DCs for CD80 and CD86 expression by
FACS, using anti-CD80 and anti-CD86 antibodies. The
results were consistent with our earlier findings; CD80


expression was reduced from 41% to 35% after LV-PLAP
infection, while CD86 expression was reduced from 61%
to 49% (Fig. 3B). Their expression was up-regulated after
transduction with LVs encoding CD80 and CD86; the


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A (I yiT nkwrti
A pTYF-LinhTlr (&---H*
V-TAR SINTLTUCliHA

pTYF-CDJ0 [ l

pTYF-CD86 C '
pTYF-FinL 0 -----
pTYF-CD40L ----- '0

pTYF-IL-12 0

pTYF-GMCSF 5
pTYF-IFN-yT
pTYF-ICAM-1 E a>O 0>


B Mock LV-PLAP LV-CD80 LV-CD86




S1714
it 9.31 ll 7.48 rtIli.'l"l1
liP ,i" 1? o .1n .-1.0 le lo,
CD80





CD86
C Mock LV-PLAP LV-CD8 LV-CD86




IFN-, ,,
---------------I FN -T1 -------------
S Mk LV LV+ILI2 LV+TCB4L





S LV+IFUf LV+IL LV+IM-CSr IV+ICAM -




IFN-T


Figure 3
LV modification of DC immune functions. (A) Diagram of LV constructs containinging different immune modulatory
genes. (B) Up-regulation of T cell costimulators in DCs transduced with LV-CD80 or LV-CD86. Immature DCs were trans-
duced with mock, LV-PLAP, LV-CD80, or LV-CD86 for 12 hr, induced to mature, and analyzed 24 hr later using anti-CD80 and
anti-CD86 antibodies. The mean fluorescence intensity and percentage of positive cells are shown. (C) Th l/Th2 assay of DCs
with up-regulated CD80 or CD86. The T-cell activation function of DCs was analyzed by DC:T cell co-culture. ICCS and FACS
for T helper function were performed 8 days after co-culture. The percentages of different T-cell populations are shown. (D)
Th I/Th2 assay of DCs co-transduced with different LV immune modulatory genes. DCs were transduced with LV (LV-PLAP),
and co-transduced with LVs encoding different immune modulatory genes, including IL-12, CD40L, IFN-y, FL, GM-CSF, and
ICAM- I, or incubated with soluble IFN-y. DCs were then treated with TNF-a and LPS and co-cultured with naive CD4 T cells.
The T cells were analyzed for IL-4 and IFN-y expression by ICCS and FACS 9 days after co-culture. The percentages of differ-
ent T cell populations are shown in the quadrants. The results are representative of six independent experiments.




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expression of CD80 was up-regulated from 35% to 44%,
and the expression of CD86 from 49% to 76%.

To see if the up-regulation of the T-cell co-stimulatory
molecules in DCs could restore the Thi response, we co-
cultured naive CD4 T cells with DCs transduced with
mock, LV-PLAP, LV-PLAP plus LV-CD80, or LV-PLAP plus
LV-CD86. After 8 days, the T cells were reactivated and
analyzed by ICCS and FACS using anti-IL-4 and anti-IFN-
y antibodies as described earlier. We found that after LV
transduction the Thl population was reduced from 24%
to 13%. Moreover, this impairment could not be cor-
rected by the up-regulation of CD80 and CD86 in DCs
(from 13% to 12% and 13%, respectively, Fig. 3C).

Modification of DC immunity by LVs encoding immune
modulatory genes
Cytokine signaling is important in DC-mediated Th differ-
entiation; for examples, IL-12 is critical to Thl develop-
ment, and Flt3-ligand (FL) has been shown to enhance IL-
12 production in DCs [30]. To overcome the impaired
Thl response after LV transduction, we investigated
whether modification of the local cytokine environment
in the DC:T cell synapse could promote a Thi response.
LVs expressing different cytokine and receptor genes,
including FL, GM-CSF, IL-12 (a bi-cistronic IL-12A and IL-
12B construct), CD40L, IFN-y, and ICAM1 were con-
structed (Fig. 3A). Expression or function of these differ-
ent immune modulatory genes has been previously
demonstrated. [21-23] DCs were transduced with LVs car-
rying reporter gene PLAP either alone or co-transduced
with different immune modulatory genes. As positive
control, we treated DCs with soluble IFN-y before matura-
tion and DC:T-cell co-culture. The Th function of the LV-
transduced DCs was analyzed by DC:T cell co-culture fol-
lowed by ICCS and FACS analysis of IFN-y and IL-4, as
described earlier. The results showed that LV transduction
alone reduced IFN-y-producing Thl cell population as
found above, from 8.16% to 3.46%. However, co-trans-
duction with LV encoding IL-12 enhanced Th response
from 3.46% to 9.38%, while co-transduction with LV
encoding IFN-y increased such response from 3.46% to
13.08%, an increase that was similar to that produced by
soluble IFN-y (Fig. 3D). LVs expressing FL, GM-CSF,
CD40L, or ICAM-1, on the other hand, exhibited no sig-
nificant effect.

Modulation of DC function by LVs expressing siRNA
targeting IL-IO
IL-10 is a critical immune modulatory gene and modula-
tion of IL-10 gene expression may alter DC function. To
test this, we constructed LVs encoding siRNA targeting IL-
10. We chose two regions in the IL-10 mRNA as the siRNA
target sites (Fig. 4A). The siRNA expression was driven by
a human H1 polIII promoter that was cloned into LVs as


previously reported. [24] The LV-siRNA vector also carries
a nlacZ reporter gene convenient for vector titer determi-
nation and for the identification of transduced cells. To
demonstrate the siRNA effects, we transduced B cells with
IL-10-siRNA LVs or a control siRNA LV targeting GFP
gene, and after transduction, the B cells were expanded
and the lacZ-positive cells were FACS-sorted using fluores-
cent substrate FDG. The expression of IL-10 was quanti-
fied by ICCS and FACS using anti-IL-10 Ab. The result
demonstrated IL-10 suppression in the lacZ-positive B
cells that were transduced with LVs expressing the two IL-
10 specific siRNAs but not the non-specific siRNA target-
ing GFP gene (Fig. 4B).

The effect of the IL-10 LV siRNAs was then examined in
DCs by co-transduction using a reporter LV and the IL-10
LV-siRNAs. The transduced DCs were then treated with
LPS and analyzed for IL-10 expression as described above.
Again, the empty LV had no effect and LV transduction
alone up-regulated IL-10 expression. However, co-trans-
duction with LV-siRNA targeting IL-10 down-regulated IL-
10 expression (Fig. 4C); the low level of IL-10 expression
in DCs was expected as the DC culture was derived and
maintained in GM-CSF and IL-4 supplemented media.

To examine whether co-transduction of DCs with LVs
expressing the IL-10 siRNA could promote a Thl
response, we transduced DCs with LV alone or together
with either an LV-siRNA (#2) or a control LV-siRNA
(GFPi). For positive control, we incubated DCs with
soluble IFN-y as previously described. After the DCs were
co-cultured with naive T cells for 10 days, the T cells were
reactivated and analyzed for Th functions by ICCS to
determine intracellular expression of IFN-y and IL-4. The
results clearly demonstrated that the IL-10 LV-siRNA vec-
tor, but not the GFPi LV-siRNA vector, enhanced Thl
response at levels comparable to that of the positive con-
trol (DCs treated with soluble IFN-y, Fig. 4D).

Discussion
Although HIV-1 is an immunopathogen in humans, HIV-
1 derived vectors do not contain viral genes and have been
rendered replication-defective. In this study, we found
that LV transduction of DCs resulted in altered DC surface
marker phenotypes. These changes in DC phenotypes led
to suppressed function in mediating the Thl immunity.
DCs transduced by LVs did not lose the capacity to stimu-
late allogeneic T-cell proliferation, as reported by others
[13,14,16]. However, in the DC:T cell co-culture func-
tional assay, we showed that after LV transduction, DCs
had significantly reduced ability to polarize naive CD4+ T
cells to differentiate into Th 1 effectors. The changed gene-
expression profile of DCs after LV transduction correlates
with Th suppression. As demonstrated here, DC-medi-
ated immunity requires antigen presentation, T cell co-


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LV siRNA


Predicted siRNA
against IL- 10:


| siI-lOH1 EFlor1 nlaZ



UUCA
5' -AGCCAUGAGTGflGUUUGAC A
1 3' -UUUCGGUACUCACUCAAACUG A G

UiCA
5' -GGGUUACCUGGGUUGCCAA A
3' -UUOCCAAUGGACCCAACGGUUA A G


P14Th
. ICS~t I -N _ICCS


IL-10


Mock


LV-GFPi LV-IL-10i LV+sIFN-y


IFN-y


Figure 4
Modification of DCs by LV-siRNA targeting IL- 10. (A) LV-siRNA targeting IL- 10. LV siRNAs targeting two different sites
of IL-10 mRNA were illustrated. The predicted hairpin siRNA structure is shown. (B) Illustration of efficient down-regulation
of IL- 10 in B lymphocytes after LV IL- 10 siRNA transduction. Epstein-Barr virus (EBV) transformed B cells were transduced
with LV siRNA targeting IL-10 (#1 and #2) or GFP gene. The siRNA LVs also carry a lacZ reporter gene which could be
labeled with fluorescein di-b-D-galactopyranoside (FDG) to separate the transduced from un-transduced cells by FACS sort.
(C) Immature DCs were transduced with mock, empty LVs, LV-nlacZ, or LV-nlacZ plus LV-silL- 10 #1 or #2, treated with LPS,
and analyzed by ICCS and FACS using anti-lL-10 antibody. (D) Enhanced ThI response by DCs transduced with LV-siRNA tar-
geting IL-10. DCs were transduced with LVs and either co-transduced with LV-siRNA targeting IL-10 (LV-ILl0i#2) or GFP
(GFPi) or treated with soluble IFN-y as controls, and the DCs were then assayed for T-cell activation function by DC:T cell co-
culture. The T cells were fully rested before reactivation with PMA and ionomycin after 10 days of co-culture. The numbers
shown in the FACS quadrants are percentages of the total gated cell population. Results are representative of three independ-
ent experiments.



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LIC"FP LII. *I L I1.IZ







http://www. retrovirology.com/content1/1 /37


stimulation, and cytokine production, all of which were
down-regulated upon LV infection. These results are con-
sistent with a recent study demonstrating cultured imma-
ture DCs and DCs from 6 of 10 HIV-1 patients display
reduced maturation function and diminished MLR in
DC:T cell coculture [28].

Cytokines have critical roles in shaping up the immune
response [31,32]. We have detected up-regulation of IL-10
in HUVEC, B cells and DCs after LV infection suggested
possible immune suppression by LVs (data not shown).
Earlier work has shown that IL-10 inhibits the expression
of IL-12 and co-stimulatory molecules in DCs,[32] a find-
ing that correlates with its ability to inhibit the primary T-
cell response and induce a stage of energy in allo- or pep-
tide-antigen-activated T cells [33]. IL-10 has also been
shown to down-regulate ICAM-1 in human melanoma
cells [34]. Here we showed that LV transduction of DCs,
led to down-regulation of CD80, CD86, and ICAM-1.
Many of these immune regulatory genes are activated
through transcriptional factor NF-KB. Using cDNA micro-
array analysis, we detected reduced NF-KB expression in
DCs after LV infection (not shown), suggesting that LV
infection may trigger a cascade of immune suppression
through down-regulation of the NF-KB signaling pathway.

It has been reported that HIV-1 Tat up-regulates IL-10 as a
result of intranuclear translocation of NF-KB and activa-
tion of the protein kinases ERK1 and ERK2 [35]. However,
the LVs used in this study do not carry a tat gene. The fact
that empty LV particles did not induce the same effects as
did intact LVs, suggests that Tat or other virion-associated
proteins do not play a role. Thus, it is plausible that events
after retroviral attachment and fusion, such as reverse
transcription and integration, might trigger the observed
cellular response. It would be interesting to see if such
immune suppression also occurs in vivo following LV gene
transfer.

DCs, during their interaction with T cells, provide multi-
ple signals to polarize naive T cells. These signals include
the co-stimulatory molecules CD80, CD86, and ICAM-1,
which are considered "signal 2" for T-cell stimulation. The
roles of these co-stimulatory molecules on Th differentia-
tion remain controversial. Many studies have shown that
ICAM-1 promotes Thl commitment [36]. CD80 and
CD86 have been reported to polarize CD4+T cells toward
the Th2 subset through engagement with CD28 [37-39].
However, CD80 could also interact with CTLA-4 to induce
Thi polarization [40]. Moreover, CD86 has been reported
to be a Thl-driving factor [41]. Further studies are needed
to address the roles of co-stimulatory molecules in the
development of DC and T-cell immunity. Nevertheless,
the down-regulation ofT cell co-stimulatory molecules in


DCs after LV transduction could potentially have an
impact on the DC-mediated Thl response.

The analysis of surface-marker expression profile also
revealed down-regulation of CDla and DC-SIGN in DCs
after LV transduction. CDla is a nonpolymorphic histo-
compatibility antigen associated, like MHC class I mole-
cules, with beta-2-microglobulin, and is responsible for
the presentation of lipid antigens. DC-SIGN (DC-specific,
ICAM-3 grabbing nonintegrin) is a 44-kDa type I mem-
brane protein with an external mannose-binding, C-type
lectin domain [42]. It has been postulated that DC-SIGN
interacts with ICAM-3 on T cells to allow sufficient DC-T
cell adhesion and, in addition, that DC-SIGN is a new
member of the co-stimulatory molecule family [5,43].
With these characteristics, the down-regulation of CDla
and DC-SIGN might also contribute to the impaired Thl
function of DCs.

Polarization of naive Th cells into Th 1 cells is critical for
the induction of cellular immunity against intracellular
pathogens and cancer cells. The observed impairment of
the Thi response by LV-transduced DCs raises a potential
issue with LV-based immunotherapy. We illustrated that
co-transduction with LV encoding IL-12 or IFN-y, but not
CD80, CD86, or ICAM-1, in DCs effectively restored Thl
immunity. In addition, co-transduction with LVs express-
ing small interfering RNA targeting IL-10 could also pro-
mote DC-mediated Thi immunity. In a step toward future
generation of vaccines, LVs encoding IL-12 and IL-10-
siRNA as potent Thl adjuvants may be used to enhance
the cellular immune response in the prime-and-boost vac-
cination regimen. In summary, our study has addressed
an important immune suppression effect of LVs and pre-
sented a solution that is important for future LV-based DC
immunotherapy applications.

List of abbreviations used
HIV-1 human immunodeficiency virus type 1

LV lentiviral vector

DC dendritic cell

Th T helper

MLV murine leukemia virus

RT-PCR reverse transcription-polymerase chain reaction

FACS fluorescence activated cell sorter

ICCS intracellular cytokine staining

IL interleukin


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PLAP placenta alkaline phosphatase

siRNA small interfering RNA

PBMC peripheral blood mononuclear cells

MOI multiplicity of infection

LPS lipopolysaccharide

TNFa tumor necrosis factor alpha

INF-y interferon-gamma


Competing interests
The authors) declare that they
interests.


have no competing


Authors' contributions
The study was conceived by LJC; XC and LJC participated
in designing and coordinating the study; JH carried out
some of the lentiviral constructions, siRNA designs and
participated in result discussion; XC performed the statis-
tical analysis; LJC and XC carried out detailed analysis of
the results and XC drafted and LJC finalized the
manuscript. All authors read and approved the final
manuscript.


Acknowledgment
We thank F. Higashikawa, W. Chou and B. Lo for technical assistance, M.
Chen and N. Benson for assistance in flow cytometry, and Dr. E. Long for
the plasmid pGEM-T-ICAM-I. We are grateful to the blood donors of Life
South. This work was supported by an NIH grant.

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