HSV-2 Infection of Dendritic Cells Amplifies a Highly
Susceptible HIV-1 Cell Target
Elena Martinelli1*, Hugo Tharinger1, Ines Frank1, James Arthos2, Michael Piatak Jr.3, Jeffrey D. Lifson3,
James Blanchard4, Agegnehu Gettie5, Melissa Robbiani1*
1Center for Biomedical Research, Population Council, New York, New York, United States of America, 2Laboratory of Immunoregulation, National Institute of Allergy and
Infectious Diseases, National Institutes of Health, Bethesda, Maryland, United States of America, 3AIDS and Cancer Virus Program, SAIC-Frederick, Inc., National Cancer
Institute, Frederick, Maryland, Unites States of America, 4Tulane National Primate Research Center, Tulane University Health Sciences Center, Covington, Louisiana, United
States of America, 5Aaron Diamond AIDS Research Center, Rockefeller University, New York, New York, Unites States of America
Herpes simplex virus type 2 (HSV-2) increases the risk of HIV-1 infection and, although several reports describe the
interaction between these two viruses, the exact mechanism for this increased susceptibility remains unclear. Dendritic cells
(DCs) at the site of entry of HSV-2 and HIV-1 contribute to viral spread in the mucosa. Specialized DCs present in the gut-
associated lymphoid tissues produce retinoic acid (RA), an important immunomodulator, able to influence HIV-1 replication
and a key mediator of integrin a4b7on lymphocytes. a4b7can be engaged by HIV-1 on the cell-surface and CD4+T cells
expressing high levels of this integrin (a4b7high) are particularly susceptible to HIV-1 infection. Herein we provide in-vivo data
in macaques showing an increased percentage of a4b7highCD4+T cells in rectal mucosa, iliac lymph nodes and blood within
6 days of rectal exposure to live (n=11), but not UV-treated (n=8), HSV-2. We found that CD11c+DCs are a major target of
HSV-2 infection in in-vitro exposed PBMCs. We determined that immature monocyte-derived DCs (moDCs) express aldehyde
dehydrogenase ALDH1A1, an enzyme essential for RA production, which increases upon HSV-2 infection. Moreover, HSV-2-
infected moDCs significantly increase a4b7expression on CD4+T lymphocytes and HIV-1 infection in DC-T cell mixtures in a
RA-dependent manner. Thus, we propose that HSV-2 modulates its microenviroment, influencing DC function, increasing
RA production capability and amplifying a a4b7highCD4+T cells. These factors may play a role in increasing the susceptibility
Citation: Martinelli E, Tharinger H, Frank I, Arthos J, Piatak M Jr, et al. (2011) HSV-2 Infection of Dendritic Cells Amplifies a Highly Susceptible HIV-1 Cell
Target. PLoS Pathog 7(6): e1002109. doi:10.1371/journal.ppat.1002109
Editor: Susan R. Ross, University of Pennsylvania School of Medicine, United States of America
Received November 11, 2010; Accepted April 23, 2011; Published June 30, 2011
This is an open-access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for
any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.
Funding: This work was supported by the NIH grant R37 AI040877, the NIH base grant RR00164, and in part with Federal funds from the National Cancer
Institute, NIH, under contract HHSN261200800001E. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: email@example.com (EM); firstname.lastname@example.org (MR)
Herpes Simplex Virus Type 2 (HSV-2) infects genital and perianal
mucosa and its infection is associated with a three-fold increased risk
of HIV-1 acquisition among both men and women . Although
active HSV-2 shedding, inflammation and ulcers during primary
infections and virus reactivation certainly contribute, their resolution
by suppressive therapy with acyclovir is not effective in reducing
HIV-1 acquisition in HSV-2 seropositive individuals . One
possible explanation for the HSV-2-driven increased risk of HIV-1
acquisition is the persistence of HSV-2-reactive CD4+T cells long
after HSV-2 replication abates . Likewise, plasmacytoid and
myeloid dendritic cells (DCs), which infiltrate areas of skin infected
therapy  and may contribute to the increased risk of HIV-1
acquisition associated with HSV-2 infection.
Epithelial cells are primary targets of HSV-2 infection. Nonetheless,
DCs, which orchestrate the immunological response to HSV-2 at its
derived DCs (moDCs) and langerhans cells found at the epithelial
infectionof DCsin-vitro hasbeen shownto inhibit theirmaturationand
immunostimulatory functions [5,6,8,9] and in-vivo HSV-2 infection
reduces HIV-1 specific T cell responses [6,10,11].
Cellular microenvironment is vital to conditioning cell function
and, in particular, the expression of receptors that affect cell
trafficking. Specialized DCs in mesenteric lymph nodes (MLNs)
and Peyer’s patches (PPs) convert vitamin A to retinoic acid (RA)
, a key factor in the control of lymphocyte trafficking and
immune responses and able to influence HIV-1 replication
[13,14,15]. In particular, RA has the unique capacity to imprint
a ‘‘gut-phenotype’’ on T cells, which includes increased expression
of integrin a4b7. The mucosal homing receptor a4b7is the
signature molecule that allows lymphocytes to gain access to the
gut tissue [16,17], a major site of HIV-1 replication . A recent
study in macaques has shown that pre-treatment with an anti-a4b7
antibody significantly reduces and delays peak plasma SIV load,
increases the percentage of CD4+T cells both in peripheral blood
and in gut tissues and reduces proviral DNA in blood and gut
tissues mononuclear cells . A specific interaction between a4b7
and the HIV-1 envelope protein, gp120, has been described 
and additional evidence shows that a4b7co-localizes with the two
main HIV-1 entry receptors CD4 and CCR5 [21,22]. The site
of gp120 binding to a4b7is conserved across gp120s from the
PLoS Pathogens | www.plospathogens.org1 June 2011 | Volume 7 | Issue 6 | e1002109
four major HIV-1 subtypes . The conserved nature of this
interaction suggests that engaging a4b7 provides a selective
advantage to HIV-1. Indeed, a4b7is expressed at high levels on
a subset of CD4+T cells that are particularly susceptible to
infection and it has been proposed that the specific affinity of HIV-
1 gp120 for a4b7provides a way for HIV-1 to target susceptible
cells at an early stage of transmission . Notably, very high
reactivity for a4b7is a characteristic shared by early transmitted
isolates, in contrast with the poor reactivity of viruses isolated
longer after transmission . Once HIV-1 crosses the epithelium,
they have to replicate at a reproductive rate R0.1 .
Expansion of the virus within the mucosa is necessary to
disseminate infection to draining LNs and blood in order to
establish a productive systemic infection. The relevance of the
HIV-a4b7interaction in the context of mucosal transmission has
yet to be elucidated.
The synthesis of RA includes two main steps. During the first
step vitamin A (retinol) is converted to retinaldehyde. This
reaction is catalyzed by a subfamily of alcohol dehydrogenases
that are expressed in most cells including DCs or by the short-
chain dehydrogenase/reductase family [12,25,26]. The second,
limiting step, is catalyzed specifically by aldehyde dehydrogenase
1A (ALDH1A) [retinal dehydrogenase] which converts retinalde-
hyde to RA. ALDH1A-expression in-vivo does not appear to be
restricted to intestinal DCs and intestinal epithelial cells as
previously suggested [25,27,28]. ALDH1A can be expressed by
LN stromal cells [29,30] and ALDH1A expressing-RA-producing
DCs are present in skin, lung and in their draining LNs .
Moreover, RA production can be induced in-vitro in splenic and
bone marrow-derived DCs (BM-DCs) . In this regard, the
granulocyte-macrophage colony-stimulating factor (GM-CSF) and
stimulation with toll-like receptor (TLR) ligands appear to play an
important role [32,33]. This raises the possibility that ALDH1A
can be expressed and induced in DCs in various tissues outside of
The present work shows that rectal HSV-2 infection of
macaques increases the percentage of a4b7highCD4+T cells in
rectal tissue and draining LNs, as well as systemically. We found
that immature moDCs express ALDH1A and this is upregulated
by HSV-2 infection. Therefore, HSV-2 increases DC’s ability to
produce RA. In turn HSV-2 infected moDCs, induce a4b7
expression expanding the a4b7highCD4+T subset through an RA-
dependent mechanism. Importantly, HSV-2 infected moDCs
increase HIV-1 replication in DC-T cell co-cultures in an RA-
dependent manner. Herein, we describe a mechanism that HSV-2
could exploit to condition its environment, influencing not only its
own replication but, possibly, that of a co-pathogen such as
Rectal HSV-2 infection of macaques increases the
percentage of a4b7highT cells
Non-human primate models constitute crucial tools to explore
mucosal infection with HIV-1/SIV, elucidate the early events after
HIV-1 transmission and how these events are impacted by other
sexually transmitted infections (STIs). We recently developed a
unique model of vaginal HSV-2 infection in rhesus macaques in
which HSV-2-infected macaques exhibited increased susceptibility
to immunodeficiency virus (simian-human immunodeficiency
virus, SHIV-RT) infection, even in absence of obvious lesions
. Building on this, we established a macaque model of HSV-2
rectal infection and we used this model to investigate early events
after HSV-2 infection across the mucosa. Clinically stable SHIV-
26108pfu of replication competent (LIVE; n=11) or UV-
inactivated (UV-HSV-2; n=8) HSV-2. UV inactivated HSV-2
(matched to the input infectious virus dose prior to inactivation)
was used to distinguish effects due to exposure to viral proteins and
nucleic acids from the effects due to viral replication. Baseline
CD4 counts and plasma SHIV RNA levels are listed for each
animal in (Table S1). Blood and rectal fluids were collected before
(BL), 3 and 6 days after challenge. Axillary, mesenteric, inguinal
and iliac LNs, as well as rectal tissue were collected following
euthanization 6 days after challenge.
HSV-2 DNA levels were measured in the rectal fluids at BL (4
days and 24 h prior to HSV-2 challenge. 24 h is shown in Table
S1) versus 3 and 6 days post-challenge by a sensitive nested HSV-2
PCR . HSV-2 shedding was detected in 9 of the 11 animals
challenged with live HSV-2 on days 3 and 6 post-challenge (Table
S1). One animal was negative on day 6 and unable to be tested on
day 3 and one was negative on day 3 and unable to be tested at
day 6. As a result, these animals were excluded from subsequent
analyses since their HSV-2 status was unclear. No DNA shedding
was detected in the animals treated with UV-HSV-2, nor in any
animals prior to challenge. Not surprisingly, CD4 counts and
plasma SHIV RNA did not show significant changes from baseline
6 days after exposure to live or UV-inactivated HSV-2 (Table S1).
Previous studies have shown that the a4b7highCD4+T cells
constitute a subset of central memory cells [21,22] and this was
similarly observed in the present study (Figure 1A). In blood of
macaques infected intra-rectally with HSV-2 we detected a
significantly higher percentage of a4b7highT cells within the
CD4+CD95+memory cell subset than before challenge and than
in macaques challenged with UV-HSV-2 (Figure 1B). The
frequency of CD4+CD95+a4b7highT cells was also higher in the
rectal mucosa of the HSV-2 infected macaques than in the UV-
HSV-2 treated ones. (Figure 1C; S1 shows the gating strategy).
The percentage of a4b7highcells in the CD4+T cell memory subset
was also significantly increased in the draining iliac LNs of HSV-2
infected macaques (Figure 1D). As expected, the gut-draining
MLNs had the highest percentage of a4b7highCD4+T cells and,
The vast majority of HIV-1 infections occur through genital
and rectal mucosa. A better understanding of the
characteristics of the mucosal microenvironment that help
HIV-1 replication is critical to developing strategies for
prevention of HIV-1 transmission. HSV-2 infects genital and
rectal mucosa and infected individuals carry an increased
risk for HIV-1 infection. Clarifying the mechanisms involved
in the increased susceptibility of HSV-2 positive individuals
to HIV-1 infection may help understating the characteris-
tics of mucosal microenvironment that facilitate HIV-1
transmission. We previously described a specific interac-
tion between HIV-1 and integrin a4b7, a signature
molecule that allows lymphocytes to gain access to the
gut tissue, a major site of HIV-1 replication. Vitamin A and
its metabolite, retinoic acid, have an important role in
balancing the immune response in the gut and in the
expression of integrin a4b7. Here we describe that HSV-2
rectal infection in monkeys increases the frequency of
a4b7+CD4+T cells in blood and rectal tissue and that this
could be at least partially explained by the ability of HSV-2
infected DCs to secrete retinoic acid and up-regulate a4b7
on CD4+T cells. These phenomena could be responsible
for increasing HIV-1 replication in DC-T cell co-cultures.
HSV-2-Infected DCs Expand a4b7high CD4+ T Cells
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together with the low levels in the distal axillary LNs, they were
unaffected by HSV-2 infection (Figure 1D). Of note, the inguinal
LNs of infected macaques also had a higher frequency of a4b7high
cells than the UV-HSV-2 treated animals. Although this difference
was not significant, finding that inguinal LNs of UV-HSV-2-
treated macaques had a significantly lower percentage of a4b7high
cells than the MLNs while this different was not significant in the
live-HSV-2 treated ones, suggests that inguinal LNs of the HSV-2
infected macaques are also enriched in a4b7highCD4+T cells
(Figure 1D). All together, these data suggest that HSV-2 infection
is either recruiting a4b7highT cells to the infected mucosa and in
draining LNs, or it is driving an up-regulation of a4b7on T cells
resident at the site of infection.
Low-level HSV-2 infection modulates moDC function
In mucosal tissues a4b7 is up-regulated on T cells by RA
produced by local DCs, therefore we set out to explore the possible
involvement of DCs in the HSV-2-driven up-regulation of a4b7in
the rectally challenged macaques. While epithelial cells are the
primary targets of HSV-2 infection, human and macaque moDCs
are susceptible to HSV-2 in-vitro [4,5,6,7]. Rationalizing that
HSV-2 is interacting with a mixed population of leukocytes in-vivo,
we used peripheral blood cells to examine which cells become
infected by HSV-2 upon exposure in-vitro. Using a flow cytometric
assay able to detect the HSV-2 early DNA binding protein ICP-8
intracellularly in infected cells, we determined that 24 h after
exposure to HSV-2 (5 MOI), on average, 0.15% [range 0.08–
0.39%] of total live cells were ICP-8+, 91.3% [range 82–99%] of
the ICP-8+were Lineage2(Lin2) and 71% [range 62–95%] of the
Lin2ICP-8+were HLA-DR+CD11c+(Figure 2B). Cell viability
was always in the range 90–95% in human PBMCs and 50–80%
in macaques and was not affected by HSV-2 infection. Although
fewer, ICP-8+CD11c+DCs were detected also after exposure to
only 0.2 MOI of HSV-2 (not shown). Therefore, CD11c+myeloid
DCs were found to be the cell subset most susceptible to HSV-2
infection in human and macaque peripheral blood mixed
leukocyte populations, supporting the possibility that HSV-2
infection of DCs plays a role in-vivo.
In order to dissect this biology more extensively, we utilized the
moDC system. Prior studies demonstrated that moDCs infected
with HSV-2 at a MOI of 5 undergo rapid apoptosis [6,34]. In order
to explore the possibility that HSV-2 infection of DCs is involved in
the up-regulation of a4b7on CD4+T on cells, we evaluated the
effect of a low-level (minimally toxic) HSV-2 infection of DCs and
how this influences DC-T cell interplay. We monitored infection of
immature moDCs exposed to varying doses of HSV-2 by ICP-8
intracellular staining and we observed a dose-dependent infection
that increased over time (Figure 3A). Annexin V/propidioum
Iodide (PI) and the LIVE/DEAD discriminator marker Aqua were
used to determine cell viability . The percentage of Annexin V2
cells was similar to the percentage of Aqua negative cells, which
represents our viable cell population. Therefore, we chose to use the
LIVE/DEAD Aqua throughout the following studies. On average
80% [range 76–85%] and 55% [range 44–58%] of the moDCs
were viable after infection with 0.2 and 1 MOI of HSV-2
(respectively), but there was little/no death of cells infected with
0.04 MOI (Figure S2). From these data we determined that the 0.2
MOI inoculum was lowest amount of virus giving reliable infection
whileleavingthe majorityofcellshealthyand abletointeract withT
cells for our DC-T cells experiments.
Figure 1. Rectal HSV-2 challenge increases the percentage of a4b7highCD3+ +CD4+ +T cells in in-vivo. A) The gating strategy for
a4b7highCD3+CD4+CD95+T cells in blood is shown from one representative animal. The majority of the a4b7highT cells are CD95+CD28+CCR7+(last plot
on the right). B) The percentages (mean 6 SEM) of CD3+CD4+CD95+T cells that are a4b7highin blood are shown at 4 days before infection (BL) and at
6 days p.i. for HSV-2-infected (LIVE, n=9) and UV-HSV-2-treated (UV, n=8) animals. C) The percentages (mean 6 SEM) of CD3+CD4+T cells that are
a4b7highin rectal mucosa are shown for HSV-2-infected (LIVE, n=7) and UV-HSV-2-treated (UV, n=5) animals 6 days p.i.. D) The percentages (mean 6
SEM) of CD3+CD4+CD95+T cells that are a4b7highin axillary (AX), mesenteric (M), inguinal (ING), and iliac LNs are shown for HSV-2-infected (LIVE, n=5)
and UV-HSV-2-treated (UV, n=7) animals. B–C) Each symbol represents an animal. (*p,0.05, **,0.01, ***p#0.001).
HSV-2-Infected DCs Expand a4b7high CD4+ T Cells
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Infections with higher doses of HSV-2 have been shown to have
immunosuppressive effects on DCs [5,6]. Therefore, changes in
DC surface phenotype and cytokine/chemokine (CK/CC) profiles
were monitored after HSV-2 infection with this lower inoculum.
There were no significant differences in the expression of the
surface markers and in the concentration of the CCs/CKs released
by moDCs 4 h after exposure to infectious (live) or UV-HSV-2
(versus mock infected DCs; data not shown). Although the
expression of CD80, CD83, CD40 and CD25 were comparable
between the differently treated groups (data not shown), the
expression of HLA-DR, CD86, CD209 and CD54 were all
significantly reduced 24 h after HSV-2 infection (Figure 3B).
HLA-DR was significantly lower in the ICP-8+(infected) fraction
of the cells exposed to infectious HSV-2, compared to the ICP-82
(undetectable uninfected or low-level infected) subset. CD86,
CD209, and CD54 were significantly reduced in the ICP-8+cells
compared to ICP-82cells within the infected population, as well as
compared to the UV-HSV-2 and mock-treated controls.
Low level HSV-2 infection of human moDCs did not alter the
release of IL1b, IL1RA, IL-5, CXCL8, IL-10, IL-12p40, IL-13,
IL-15, IL-17, IFNa, IFNc, CCL2, CCL3, CCL4, CXCL9,
CCL11, CCL5 (data not shown). However, the levels of IL-2,
IL-2R, IL-6, IL-7, TNFa, and CXCL10 were significantly
increased in the HSV-2 infected DC cultures (Figure 3C).
Notably, the amount of CXCL10 in the supernatants of HSV-2
infected DCs was, on average, 25 fold [95%CI 5–45] higher than
both UV-HSV-2 and mock-infected cells. Overall, these results
indicate that the infection of moDCs with 0.2 MOI of HSV-2
minimizes apoptosis in the first 24 h post infection (p.i.), retaining
the ability of HSV-2 infection to modulate DC phenotype and
function. This allowed us to study how the HSV-2 driven changes
on DCs influence the DC-T interplay.
HSV-2 infected moDCs induce a4b7on CD4+T cells via RA
CD4+T cells cultured for 5 days with autologous DCs can be
divided in 4 subsets on the basis of their expression of a4b7. While
a4b7lowT cells (LOW) are naı ¨ve T cells, a4b7int(INT), a4b7high
(HIGH) and a4b7negpresent a memory phenotype (Figure 4A and
S3). After 5 (and 7, not shown) days of culture, the percentage of
a4b7highT cells was significantly increased in HSV-2-infected DC-
T cell co-cultures (Figure 4A and B), while there was no significant
difference in the a4b7intand a4b7low(not shown). In contrast,
CD69 expression was significantly reduced in co-cultures with
HSV-2-infected DCs, (relative to the mock and UV-treated cells;
Figure 4A and C). It has been shown that the expression of a4b7
on naı ¨ve T cells is enhanced upon antigenic stimulation with
MLN-DCs and PP-DCs . Therefore, the small, non-significant
increase in a4b7expression seen in the UV-HSV-2 condition could
be explained by stimulation of TLRs due to HSV-2 proteins and
nucleic acids, the amount of which would be increased in cultures
with replicating virus. However, even treatment of moDCs with 25
fold more UV-HSV-2 (equivalent to an MOI of 5), did not
significantly alter the a4b7expression on the CD4+T cells, but
increased CD69 expression even further (Figure S4 A and B).
T cells cultured alongside in the absence of DCs, but in the
same amounts of live-HSV-2 used to treat the DCs (0.2 MOI), did
not show evidence of infection (Figure S5 A), alteration of viability
status (not shown) or changes in the expression of a4b7(Figure S5
B) 5 days p.i. However, as previously reported , T cells treated
with a higher inoculum are susceptible to HSV-2 infection, as
demonstrated by the detection of ICP-8+CD4+T cells after
exposure to 5 MOI of HSV-2 (Figure S5 A). T cell viability and
expression of a4b7 remained unchanged after exposure to the
higher dose of HSV-2 (Figure S5 B). On the contrary, expression
of CD69 was significantly up-regulated on the T cells 5 days post
HSV-2 infection both in cells infected with 0.2 and 5 MOI
(Figure S5 C).
Since RA is known to increase a4b7expression in T cells, a
selective RA receptor (RARa) antagonist  was included in the co-
cultures. The RARa antagonist blocked the HSV-2-infected DC-
induced up-regulation of a4b7on the T cells (Figure 4B). The slight
increase in a4b7highcells in the UV-HSV-2-treated compared to
mock-infected DC-T cell co-cultures was also blocked (Figure 4B).
Figure 2. Blood CD11c+ +DCs are susceptible to HSV-2
infection. A) Human (n=3) and macaque (n=3) PBMCs were HSV-2-
infected (5 MOI, LIVE) or mock-treated and the ICP-8 expression
measured 24 h later. ICP-8 versus CD11c expression is shown for
Lin2HLA-DR+cells on representative examples. The percentages of the
positively stained cells are provided. B) The percentages (mean 6 SEM
of 3 donors each) of total live PBMCs that are ICP8+, of ICP8+cells that
are Lin2, and of the ICP8+Lin2cells that are HLA-DR+CD11c+are shown
for human (HU) and macaque (RM) cultures.
HSV-2-Infected DCs Expand a4b7high CD4+ T Cells
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Figure 3. Low level HSV-2 infection modulates DC function. A) Immature moDCs were exposed to 0.04, 0.2, or 1 MOI of replication competent
HSV-2 (or medium, MOCK) and infection was monitored by measuring the expression of ICP-8 at 4 h and 24 h. The percentage of ICP-8+(infected)
cells is indicated in each panel. One representative experiment of 5 is shown. B) DCs were treated with 0.2 MOI of replication competent (LIVE) or UV-
HSV-2 (UV) versus mock (MOCK) supernatant and the surface phenotype assessed after 24 h. The mean fluorescent intensities (MFI) (means 6 SEM, 5
HSV-2-Infected DCs Expand a4b7high CD4+ T Cells
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co-cultures, although its effect was much less pronounced than in the
HSV-2 infected and UV-HSV-2 DC-T cell co-cultures (Figure 4B).
Of note, CD4+T cells cultured for 5 days in absence of moDCs, but
in the same culturing conditions as the DC-T cell co-cultures,
expressed a lower level of a4b7than in presenceof mock, UV-HSV-2
(Figure 4B).The RARa antagonist did not alter the CD69 expression
(Figure 4C), suggesting that its effect was highly specific, did not
generically alter the viability status of the cells, and that RA is not
involved in the reduced CD69 expression by the T cells. These data
demonstrate that RA is at least partially responsible for inducing
expression of a4b7on the CD4+T cells.
HSV-2 infection increases ALDH1A1 expression in moDCs
The irreversible enzymatic conversion of retinaldehyde to all-
trans-RA by ALDH1A [RALDH] constitutes the limiting step in
the production of metabolically active RA. There are 3 main
Figure 4. HSV-2 infection of moDCs induces a4b7on CD4+ +T cells. Mock-, UV-HSV-2-, or Live HSV-2-treated DCs (Figure 3) were mixed with
autologous CD4+T cells and cultured for 5 days. A) The gating strategy for the definition of the a4b7high(HIGH) (memory cells), a4b7int(INT) (memory
cells) and a4b7low(LOW) (naı ¨ve cells) and CD69+subsets is shown for 1 representative of 11 independent experiments. B–C) The fold changes (mean
6 SEM, 11 independent experiments) in the percentage of a4b7highCD3+CD4+T cells (B) and CD69+CD3+CD4+T cells (C) in the absence (2) or
presence (+) of an RARa antagonist are shown relative to the mock-treated controls (set as 1). A T cell only control (no DCs, T ONLY), not exposed to
HSV-2, was included alongside. (*p,0.05, **,0.01, ***p#0.001).
independent experiments) of the indicated markers are shown for the total mock (MOCK) and total UV-HSV-2-treated (UV) DCs versus the ICP-82(2)
and ICP-8+(+) fractions of the LIVE condition. C) CK/CC concentrations (means 6 SEM, 11 independent experiments) in the cell-free supernatants
from LIVE-, UV-, and MOCK-treated moDCs are shown. (*p,0.05, **,0.01, ***p#0.001).
HSV-2-Infected DCs Expand a4b7high CD4+ T Cells
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isoforms of ALDH1A. ALDH1A1 [RALDH1] is expressed by
DCs in PPs and by epithelial cells of the intestine, while
ALDH1A2 [RALDH2] is expressed in MLNs . ALDH1A3
is expressed at much lower levels in both PPs and MLNs.
Peripheral LNs express ALDH1A2 but at a barely detectable level
. Murine BM-DCs, differentiated in GM-CSF and IL-4,
express ALDH1A2 . We investigated whether human moDCs
express ALDH1A and if the expression of a4b7on T cells in the
DC-T cell co-cultures could be related to a de-novo production of
RA by the DCs.
We found that immature moDCs express ALDH1A1 and
ALDH1A2, but not ALDH1A3 (Figure 5A). A relative quantifi-
cation of the expression levels by RT-qPCR indicated that HSV-2
infection significantly increased the expression of ALDH1A1
compared to the UV-HSV-2- and mock-treated controls (Figure
5B). It has been shown that TLR ligands enhance ALDH1A2
expression in BM-DCs . However, although the UV-HSV-2-
treated moDCs expressed higher level of ALDH1A1 compared
with the mock-treated cells, this difference was not significant.
Moreover, the treatment with 25-fold more UV-HSV-2 did not
significantly up-regulate ALDH1A1 in immature moDCs (Figure
S6 A). We also found that generic aldehyde dehydrogenase activity
(ALDH) increases significantly in HSV-2-infected moDCs com-
pared with the UV-HSV-2 treated and mock-treated controls
(Figure 5C and D). Because the assay measuring ALDH activity
could not be used in combination with ICP-8 intracellular staining
we could not distinguish if the increased ALDH activity was
restricted to the HSV-2 infected cells, or if there was contribution
from bystander non-infected cells. Thus, these data indicate that
human moDCs have the potential to produce RA and that
replication competent HSV-2 induces increased expression of the
rate-limiting enzyme that converts retinaldehyde to RA.
HSV-2 infection enhances HIV-1 replication in a
Previous studies showed that RA profoundly affects HIV-1
replication in-vitro [13,14,15]. Having shown that HSV-2-infected
DCs increase the percentage of a4b7highT cells through an RA-
dependent mechanism, we evaluated the impact of HSV-2
infection of immature moDCs on HIV-1 replication in DC-T cell
mixtures. HSV-2-infected (versus UV-HSV-2- and mock-treated
DCs) were pulsed with the CCR-5 tropic HIV-1 ADA-M,
extensively washed and then mixed with autologous CD4+T
cells. HSV-2 infection of DCs significantly increased HIV-1
infection in CD4+T cells in the DC-T cell co-cultures (Figure 6A–
C). Although there was high variability in the level of HIV-1
infection and enhancement from donor to donor, the HSV-2-
driven increase was consistent and in 1 out of 15 experiments
reached a 100-fold increase compared to the mock condition. We
found a modest increase in HIV-1 replication of UV-HSV-2-
treated DC-T cell co-cultures compared with the mock condition,
but this was not statistically significant when data from multiple
donors was evaluated (Figure 6B). However, higher amounts of
UV-HSV-2 induced higher HIV-1 replication (Figure S6 B),
paralleling the increased CD69 expression seen in these cultures
(Figure 4C and S4 B).
As previously suggested [21,22], the a4b7highCD4+T cells are
highly susceptibleto HIV-1 infection, as evidenced by p24 expression
in the various T cell subsets (Figure 6C). Specifically, the percentage
of p24+cells in the a4b7highsubset was always higher than in the
a4b7intsubset [1.6 fold, 95%CI: 1.1–2.1], than in the a4b7lowsubset
[4.8 fold, 95%CI: 1.5–8.2] and than in the a4b7negsubset [2.8 fold,
95%CI: 2.4–3.2] (Figure 6C). In all experiments, the greater
difference between the a4b7highand the other subsets was found in
the HSV-2-infected DC-containing cultures.
Since T cells can be infected with HSV-2 (Figure S5 A and
), we examined the effect of HSV-2 infection of CD4+T cells
on HIV-1 replication (in absence of DCs). We found a small
increase in HIV-1 replication in the CD4+T cells treated with
replication competent HSV-2 compared to the mock condition
similar to the increase in the UV control (Figure S7 A), coincident
Figure 5. HSV-2 infection increases ALDH1A1 expression in
moDCs. A) Expression of ALDH1A1, ALDH1A2, and ALDH1A3 RNA in
immature moDCs and PBMCs by RT-PCR. Results shown are represen-
tative of data from 2 different donors. A negative control (2) without
DNA was included to control for contamination. GAPDH was amplified
in each sample as an internal PCR and loading control. B) The fold
changes (mean 6 SEM, 13 independent experiments) in ALDH1A1
mRNA of LIVE- and UV-HSV-2-treated moDCs (24 h post treatment) are
shown relative to mock-treated moDCs (set as 1). C) The fold changes
(mean 6 SEM, 5 independent experiments) in the percentage of
ALDEFLUOR positive (ALDH enzymatic activity) moDCs treated with
LIVE or UV-HSV-2 (24 h post treatment) are shown relative to mock-
treated moDCs (set as 1). D) Gating strategy for ALDH enzymatic activity
in mock-, UV-HSV-2-, or Live HSV-2-treated moDCs (summarized in
panel C). One representative of 5 independent experiments is shown.
The numbers indicate the percentage of ALDH+cells. (*p,0.05,
HSV-2-Infected DCs Expand a4b7high CD4+ T Cells
PLoS Pathogens | www.plospathogens.org7June 2011 | Volume 7 | Issue 6 | e1002109
with the increased CD69 expression within these cultures (Figure
Next we tested the effect of the RARa antagonist on the HIV-1
infection in the DC-T cell cultures. Inclusion of the RARa
antagonist blocked the increased HIV-1 replication seen in the
presence of HSV-2-infected DCs, but had low or no impact on the
HIV-1 replication in the mock- and UV-HSV-2-treated DC-T cell
mixtures (Figure 6D). With the exception of the experiment
exhibiting 100-fold increase in HIV-1 replication, the RARa
antagonist was able to inhibit infection to a level similar to that of
the mock DC-T co-cultures (Figure 6D). The RARa antagonist
had no effect on low-level HIV infection of CD4+T cells cultured
in the absence of DCs (Figure S7 B). These data confirm that
HSV-2-infection of DCs augments HIV-1 replication in DC-T cell
mixtures via an RA-dependent pathway.
HSV-2 enhances HIV-1 acquisition and transmission during
symptomatic and asymptomatic stages of HSV-2 infection .
However, due to the lack of a suitable animal model for HSV-2
infection that closely relates to humans, the underling mecha-
nism(s) that leads to enhanced risk of HIV-1 infection remains
unknown. One potential explanation holds that increased presence
and persistence of HSV-2-reactive CD4+T cells facilitate HIV-1
Herein, we provide in-vivo data collected in a novel non-human
primate model of HSV-2 infection and we describe in-vitro
experiments that add new insights into HSV-2/HIV-1 interplay.
In-vivo we observed an increase in the percentages of a4b7highCD4+
T cells both locally and systemically a few days after rectal HSV-2
challenge. We show that CD11c+DCs from peripheral blood are
susceptible to HSV-2 infection in-vitro and that HSV-2 infection of
immature moDCs amplifies the a4b7highCD4+T subset in
autologous DC-T co-cultures. We show that HSV-2 infection
increases ALDH1A1 expression in DCs, a phenomenon that
enhances their potential to produce RA. The latter mediates the
HSV-2-driven up-regulation of a4b7in our DC-T co-cultures and
has a plethora of immunomodulatory effects, including influencing
HIV-1 replication [13,15] Indeed, we found that blocking the
RARa in T cells inhibits HIV-1 replication in HSV-2-infected
DC-T cell cultures.
Localization, retention, function and survival of antigen-
experienced T cells that infiltrate mucosal sites following pathogen
invasion, are influenced by the expression of adhesion molecules,
substantially modulated by microenvironmental factors .
Among such adhesion molecules, the integrin receptor a4b7
mediates lymphocyte migration to the gastrointestinal tract.
However, recent findings indicate that STIs can modulate the
expression and migration of a4b7+lymphocytes also in other
tissues, such as the endocervix of human females infected with
Clamydia trachomatis [39,40]. We developed a macaque HSV-2
rectal infection model and show that in macaques the mucosal site
of HSV-2 infection, its draining LNs, and blood are enriched in
a4b7highT cells within 6 days of HSV-2 exposure. The increased
percentages of a4b7highT cells were not observed in animals
treated with UV-HSV-2, suggesting that HSV-2 replication is
important to this phenomenon.
Several factors could explain the enrichment in a4b7highT cells at
the site of HSV-2 infection. Among them is the ability of a4b7highT
cells to specifically target mucosal sites, a possible inflammation-
driven induction of the a4b7receptor MadCam [41,42], and specific
responses of CM T cells to inflammatory soluble factors. However,
DCs are present and persist at the site of HSV-2 infection , they
are critical to the immunological response to HSV-2 [43,44,45] and
are able, in determinate circumstances, to induce a4b7on T cells.
Therefore, we explored the possibility of their contribution to the
HSV-2 is able to skew DC immunological responses [5,6,46].
While moDCs and langerhans cells are highly susceptible to HSV
in-vitro [4,5,6,7], plasmacytoid DCs, critical players in the innate
response to HSV , seem to be resistant to infection .
CD11c+myeloid DCs are important in antigen presentation and
adaptive response to HSV . Mimicking the mixed leukocyte
populations potentially encountering HSV-2 in-vivo using blood,
we confirmed that (macaque and human) myeloid CD11c+DCs
are the primary leukocyte target for HSV-2 infection in-vitro. Due
to the variety of CD11c+DC subsets implicated at different stages
of the immune response  future studies will need to investigate
the precise phenotype of the susceptible population, the differences
between HSV-2-infected in-vitro generated moDCs, and infected
CD11c+DCs in their interaction with T cells. Since the primary
goal of this study was to explore whether modulation of myeloid
DC function by HSV-2 infection was involved in the enrichment
of a4b7highT cells observed in-vivo, we were able to use the
established moDC-HSV-2 model to dissect this biology.
Figure 6. HSV-2 infection enhances HIV-1 replication in a RA-
dependent manner. Mock-, UV-HSV-2-, or Live HSV-2-treated DCs
were loaded with HIV-1 and co-cultured with autologous CD4+T cells
for 3 to 7 days (A) or 5 days (B–E). A) The kinetics of infection is shown
for 1 of 3 similar experiments. B) The fold changes (mean 6 SEM of 15
independent experiments) in HIV-1 DNA copies/cell for the 3 conditions
after 5 days of co-culture are shown relative to the MOCK controls (set
as 1). C) The percentage of p24+CD4+T cells within each a4b7subset
was measured 5 days after co-culture with the differently treated DCs. 1
of 3 independent experiments is shown. D) The fold changes (mean 6
SEM, 4 independent experiments) in the HIV-1 copies/cell in presence
(+) or absence (2) of the RARa antagonist are shown relative to the
mock-treated controls (set as 1). One experiment, in which the HIV-1
infection was about 100 fold higher in the live HSV-2 condition
compared to the mock treated, was excluded. In the excluded
experiment the RARa antagonist did not reverse the HSV-2-infected
DC enhancement effect (reduced by 20%). (*p,0.05, **,0.01,
HSV-2-Infected DCs Expand a4b7high CD4+ T Cells
PLoS Pathogens | www.plospathogens.org8 June 2011 | Volume 7 | Issue 6 | e1002109
The effect of HSV-2 infection on moDCs has been extensively
studied, typically using relatively large amounts of virus [4,5,6,7].
Our work reveals that even a much smaller viral inoculum
significantly influences DC biology. We confirmed that low dose
HSV-2 infection caused a down-regulation of the maturation
receptors HLA-DR, CD86 and CD54, as seen with higher HSV-2
doses [49,50]. We also demonstrated a down-regulation of
CD209, which would be expected in a maturing DC. The latter
can be explained by the ability of HSV to bind this receptor ,
although it could be also the result of a skewed maturation process.
We demonstrated that DCs infected with a low HSV-2 inoculum
down-modulated CD69 expression on T cells, supporting an
earlier report that HSV-infected moDCs inhibit T cell activation.
Notably, we found that HSV-2-infected DCs up-regulate the
expression of a4b7and, by blocking the binding of RA to its
receptors on the CD4+T cells, we showed that RA was directly
involved in the HSV-2 driven increase in a4b7expression. RA
impacts several immunological mechanisms, in particular it is
known to induce a mucosal-type phenotype in DCs , playing
an important role in inducing and sustaining the tolerogenic
microenvironment of the gut .
We provide the first evidence that human immature moDCs
express ALDH1A1 (and ALDH1A2), have the potential to convert
serum retinol into RA, and that HSV-2 infection significantly
increases this capability. This supports earlier work in mice
showing that GM-CSF and IL-4 induce ALDH1A2 expression in
BM-DCs . The same study also reported that this gene was
up-regulated by TLR ligands in DCs cultured with GM-CSF and
IL-4 and matured with LPS. However, the up-regulation of
ALDH1A1 expression by HSV-2 infection in human moDCs did
not appear to be due a ligand effect of HSV-2 proteins or DNA
(triggering through TLRs), since even 25-times more UV-HSV-2
was unable to reproduce these responses. Additional studies are
needed in order to ascertain whether other TLR ligands or other
pathogens can stimulate human DCs (like HSV-2 infection) to up-
regulate ALDH1A1 expression and subsequently increase a4b7
expression on CD4+T cells. The specific mechanism through
which HSV-2 infection increases ALDH1A1 expression in moDCs
was not a major focus of this work and might be a direct effect of
newly synthesized HSV-2 components and/or an indirect effect of
CCs/CKs secreted by DCs in response to HSV-2 replication.
Given the potentially important role that RA holds in immune
responses to pathogens, this subject is worthy of further research.
That HSV-2 is able to mediate the up-regulation of an enzyme
that serves as a key metabolic checkpoint in the conversion of
retinol to RA is noteworthy, because RA has the capacity to
modulate immune responses and replication of many pathogens
including HIV-1 [15,25,52].
These studies also revealed that significantly elevated amounts
of other soluble factors are released by HSV-2-infected moDCs. In
particular, we detected a notable increase in IL-7, which is known
to induce HIV-1 reactivation and replication in T cells [53,54]
and, as previously reported , of CXCL10 which is responsible
of recruiting activated T cells, therefore contributing to viral
replication in inflamed tissues . All these factors could
cooperate in enhancing HIV-1 infection. However, an RAR
antagonist ablated the HSV-2-mediated enhancement of HIV-1
amplification, suggesting that RA is one of the major factors
driving this biology.
HIV-1 infection of moDCs could also be affected by HSV-2
infection. Though, the apoptotic nature of the HSV-2 infection,
suggests little contribution of HIV-1 replication in moDCs to the
enhanced HIV-1 replication in the co-cultures.
We previously reported that a4b7highT cells are the most
susceptible HIV-1 target in T cells cultures supplemented with RA
and that blocking a4b7 binding to HIV-1 inhibits HIV-1
replication [20,21]. Herein, we show that the a4b7highT cells also
constitute the most susceptible HIV-1 target in the DC-T cell co-
cultures and that this is independent of the effect of HSV-2 on the
DCs. Therefore, being particularly susceptible to HIV seems an
intrinsic characteristic of a4b7highCD4+T and an expansion of this
cell-subset likely has a greater impact than the expansion of less
susceptible subsets, contributing to fuel infection.
This work gives us new insights into HSV-2 modulation of the
mucosal microenvironment. A low- level HSV-2 infection of
immature myeloid DCs could play a role in increasing the
susceptibility to HIV-1 by influencing its surroundings in a way
favorable to HIV-1 infection. In Figure 7 we try to integrate our
findings in a bigger picture with the new different actors that
HSV-2 infected DCs add to the scene. Further studies will have to
dissect how these mechanisms interplay in-vivo, the respective role
of factors such as RA and a4b7and their relative importance in
transmission across the rectal and genital mucosa.
Materials and Methods
Adult female Chinese rhesus macaques (Macaca mulatta) were
housed and cared for in compliance with the regulations under the
Animal Welfare Act, the Guide for the Care and Use of Laboratory
Animals, at Tulane National Primate Research Center (TNPRC;
Covington, LA). Animals were monitored continuously by veteri-
narians to ensure their welfare. Veterinarians at the TNPRC
Division of Veterinary Medicine have established procedures to
Figure 7. Potential mechanism of HSV-2-infected DCs enhance-
ment of HIV-1 infection. Rapid replication of HSV-2 in epithelial cells
could drive a low level HSV-2 infection in neighboring DCs, imprinting
on them a ‘‘mucosal-like’’ phenotype and inducing the ability to release
RA. RA can profoundly affect resident and/or recruited lymphocytes and
expand the pool of a4b7highCD4+T cells at the mucosal site of infection.
These highly susceptible cells could contribute to increase HIV-1
replication rate and HIV-1 rapid access gut inductive sites, PPs and
MLNs. Therefore, the RA-driven immunomodulatory effect and other
HSV-2 driven changes – that need to be investigated - could partner to
create an environment particularly susceptible to HIV-1 infection.
HSV-2-Infected DCs Expand a4b7high CD4+ T Cells
PLoS Pathogens | www.plospathogens.org9 June 2011 | Volume 7 | Issue 6 | e1002109
minimize pain and distress through several means. Monkeys were
anesthetized with ketamine-HCl (10 mg/kg) or tiletamine/zolaze-
pam (6 mg/kg) prior to all procedures. Preemptive and post
procedural analgesia (buprenorphine 0.01 mg/kg) was required for
procedures that would likely cause more than momentary pain or
distress in humans undergoing the same procedures. The above
listed anesthetics and analgesics were used to minimize pain or
distress associated with this study in accordance with the
recommendations of the Weatherall Report. Any sick animals were
euthanized using methods consistent with recommendations of the
American Veterinary Medical Association (AVMA) Panel on
Euthanasia. All studies were approved by the Animal Care and
Use Committee of the TNPRC (OLAW assurance #A4499-01) and
in compliance with animal care procedures. TNPRC is accredited
by the Association for Assessment and Accreditation of Laboratory
Animal Care (AAALAC#000594).
Cells and reagents
Immature moDCs were generated as previously described .
Briefly: Peripheral blood mononuclear cells (PBMCs) were isolated
from heparinized human leukopacks (New York Blood Center,
New York, NY) using Ficoll-Hypaque density gradient centrifu-
gation (Amersham Pharmacia Biotech, Uppsala, Sweden). CD14+
monocytes were isolated using the human CD14 magnetic cell
sorting (MACS) system (Miltenyi Biotec, Auburn, CA) and moDCs
generated by culturing these cells in 100 U/mL recombinant
human interleukin-4 (IL-4) (R&D Systems, Minneapolis, MN) and
1000 U/mL recombinant human granulocyte-macrophage colo-
ny-stimulating factor (GM-CSF) (Berlex Laboratories, Montville,
NJ). After 5 days, immature moDCs were collected, an aliquot
taken for flow cytometric analysis (BD FACSCAlibur) of DC
phenotype and maturation [FITC anti–HLA-DR, APC anti-
CD25, PE anti-CD80, PE anti-CD83, anti-CD86, PE anti-CD3
(see later for clones)]. moDC purity was greater than 98%. Cells
were negative for CD80 and CD25; less than 1% of moDCs
expressed CD83. The remainder of the cells was used for HSV-2
infections. moDCs were cultured in R1 [RPMI 1640 (Cellgro;
Fisher Scientific, Springfield, NJ) containing 2 mM L-glutamine
(GIBCO Life Technologies, Grand Island, NY), 10 mM HEPES
GIBCO Life Technologies), 50 mM 2-mercaptoethanol (Sigma
Chemical, St Louis, MO), penicillin (100 U/mL)/streptomycin
(100 mg/mL) (GIBCO Life Technologies), and 1% heparinized
human plasma]. Autologous CD142cells were cultured 5 days in
R10 [RPMI 1640 with 2 mM L-glutamine, 10 mM HEPES,
50 mM 2-mercaptoethanol, penicillin 100 U/mL/streptomycin
and 10% of FBS] supplemented with 1 U/ml of IL2 (Preclinical
Repository, National Cancer Institute at Frederick, NCI-Freder-
ick, MD) at 206106cells/ml. The day before starting the DC-T
culture, day 5, CD4+T cells were isolated using the human CD4
positive MACS system (Miltenyi). CD4+T cells were cultured
overnight in R5 [RPMI 1640 with 2 mM L-glutamine, 10 mM
HEPES, 50 mM 2-mercaptoethanol, penicillin 100 U/mL/strep-
tomycin and 5% of human AB serum (Sigma-Aldrich)] supple-
mented with 1 U/ml of IL2, at 106106cells/ml.
Viral stocks were propagated in Vero cells (American Type
Culture Collection [ATCC] Manassas, VA), titered by plaque
formation on Vero cells, and aliquots stored at 280uC . HSV-
2 was inactivated by exposure to UV lamp for 6 h in 6 wells plates
without lid. Inactivation was verified by plaque formation on Vero
cells. Freshly isolated human and macaque PBMCs were
resuspended in cold RPMI 1640 at 506106cells/ml and exposed
to 0.2 or 5 plaque forming units (pfu)/cell (1 MOI=1 pfu/cell)
of live or UV-inactivated HSV-2 or to the equivalent volume of
medium in which the viral stocks were grown (mock) (Dulbecco
modified Eagle medium [DMEM] 2% FBS) for 2 h at 37uC cells
were extensively washed and cultured for 24 h in R10. 1 U/ml of
IL2 was added to the PBMC cultures.
Immature moDCs were collected at day 5, resuspended in cold
RPMI 1640 without FBS at 206106cells/ml, exposed to live 2
(0.04, 0.2 or 1 pfu/cell) or UV-inactivated HSV-2 (0.2, 1 or 5 pfu/
cell) or to DMEM (mock-treated) for 2 h at 37uC. Cells were
extensively washed and cultured for 24 h in R1 (with 100 U/mL
IL-4and 1000 U/mL GM-CSF)at 16106cells/ml in 6 wells plates.
HSV-2 infection was confirmed at 4 and 24 h by flow cytometry
(BD FACSCAlibur). Although not a perfect control, 5 pfu/cell of
UV-HSV-2 (25 fold the live inoculum) was rationalized from the
infection of Vero cells. These cells release a maximum amount of
virus corresponding to 16 folds their inoculum after 24 h in culture.
PBMC and moDC immunostaining
HSV-2-infected PBMCs were washed and resuspended in PBS
with LIVE/DEAD Fixable Aqua (Invitrogen, Life Technologies)
for 10 mins at 4uC. Cells were washed with PBS, resuspended in
FACS wash buffer (PBS 5% BSA 0.1% Na Azide) and incubated
for 30 mins at 4uC with: Pacific Blue anti-CD3 (clone SP34-2) and
anti-CD14 (clone M5E2), Alexa700 anti-CD20 (clone 2H7), PCP-
Cy5.5 anti-HLA-DR (clone L243), PeCy7 anti-CD11c (clone 3.9).
Cells were washed with FACS wash and fixed/permeabilized with
Fix/Perm and Wash/Perm buffers (BD Biosciences). Cells were
stained with anti-HSV-2 ICP-8 monoclonal antibody (mAb)
(IgG2a isotype; Virusys, North Berwick, ME) 15 mins at room
temperature, washed and analyzed within 24 h with BD LSRII.
The ICP-8 mAb was directly conjugated with Alexa647 (Zenon
Antibody labeling kit, Invitrogen, Life Technologies). Data were
analyzed with FlowJo software 8.8.6.
HSV-2-infected and mock-treated moDCs were washed and
resuspended in PBS with LIVE/DEAD Fixable Aqua for 10 mins
at 4uC. Cells were washed with PBS, resuspended in FACS wash
buffer incubated 30 mins at 4uC with FITC anti–HLA-DR (clone
L243), PE anti-CD25 (M-A251), PE anti-CD80 (clone L307.4), PE
anti-CD86 (clone), PE anti-CD209 (clone DCN46), PE anti-CD40
(clone 5C3), PE anti-CD54 (clone HA58) PE anti-CD83 (clone
HB15e) (all BD Biosciences). Cells were fixed and permeabilized
with Fix/Perm and Wash/Perm buffers (BD Biosciences). Cells
were stained with anti–HSV-2 ICP-8 mAb 15 mins at room
temperature, washed and analyzed within 24 h with BD FACS
Calibur or with BD LSRII, when stained with Aqua.
DC-T cell assays
HSV-2-infected (0.2 MOI) versus UV-HSV-2- and mock-
treated DCs were resuspended in PBS 1% BSA at 106106cells/ml
and exposed to 86104TCID50/106cells of HIV-1 ADA-M (Lot:
P4023. Sucrose gradient-purified was kindly provided by the AIDS
Vaccine Program, SAIC-Frederick, NCI-Frederick) for 2 h at
37uC (versus no virus controls as indicated). 30 mins before the
end of the 2 h, CD4+T cells were resuspended at 66106cells/ml
in R5 supplemented with 1 U/ml of IL-2 and plated in 48 well
plates. RAR antagonist Ro41-5253 (Enzo Life Sciences, Zandho-
ven Belgium) at the final concentration of 500 nM or the same
quantity of dimethyl sulfoxide (DMSO) was added to the wells for
10 mins at room temperature. HIV-1-pulsed moDCs were washed
3 times and resuspended at 26106cells/ml in R5 (1 U/ml IL2).
Cells were mixed at a 1:3 ratio in 48 well plates (0.56106DC:
1.56106T cells) and cultured at a final concentration of 46106
cells/ml. Control CD4+T cells were cultured at 46106cells/ml in
R5 (1 U/ml IL2), exposed to live, UV HSV-2 or DMEM and/or
HSV-2-Infected DCs Expand a4b7high CD4+ T Cells
PLoS Pathogens | www.plospathogens.org10 June 2011 | Volume 7 | Issue 6 | e1002109
co-exposed to 86104TCID50/106cells of HIV-1 ADA-M (Lot:
P4023) for up to 5 days. Viruses were added directly to the CD4+
T cells cultured in absence of moDCs and not washed out. After 3,
5 and 7 days pellets and supernatants from the HIV-infected DC-
T cell and T cell only cultures were collected and stored at 280uC.
HIV-naive DC-T cell and T cell only cultures were stained with
LIVE/DEAD Fixable Aqua (Invitrogen, Life Technologies),
PerCP-Cy5.5 anti-CD4 (clone SP34-2), Pacific Blue anti-CD3
(clone L200), Alexa700 anti-CD69 (Clone FN50), FITC anti-
CD45RA (clone HI100), APC anti-CD45RO (clone UCHL1),
FITC anti-CD62L (clone SK11), PeCy7 anti-CCR7 (clone 3D12),
PE anti-dimeric a4b7 (Act-1 Clone, NIH AIDS Research
Reference and Reagent Program). Act-1 was directly conjugated
using LYNX RPE antibody Conjugation KIT (AbD Serotech,
Raleigh, NC) at 4uC for 30 mins. Cells were fixed in Cytofix buffer
(BD Bioscience). At least 200000 events in the lymphocyte gate
were acquired and analyzed using the BD LSRII Flow Cytometer
and the FlowJo 8.8.6 software (Tree Star, Inc.).
DNA from DC-T cell pellets was extracted using DNeasy Blood
& Tissue kit (Qiagen Inc, Valencia, CA). Quantitative PCR for
HIV gag DNA and an estimation of cell numbers using albumin
DNA copy numbers was performed using published primers and
molecular probes . DNA was quantified by qPCR with an ABI
7000 PCR machine (PerkinElmer Life and Analytical Sciences,
Boston, MA) using 5 ml of DNA for 40 cycles and the ABI master
mix (TaqMan Universal PCR Master Mix; Applied Biosystems).
ALDH1A RT- qPCR
ALDH1A Primers are listed in Supplementary Table S2 and
were generated using Primers3 . They were designed to span at
least 1 exon-exon junction and their specificity was verified by
nucleotide blast. The GAPDH gene was used as positive assay
control with primer sequences: FW GAAGGTGAAGGTCG-
GAGT, RW GAAGATGGTGATGGGATTTC. RNA extraction
was carried out using RNeasy Mini Kit (Quiagen) and residual
DNAwasdigested usingtheRNase-Free DNase Set(Quiagen). The
reverse transcription (RT) was performed using RandomPrimers
(Invitrogen), dNTPmix (BioRad) DTT (5 mM, Invitrogen) Super-
script III RT and 56 First strand buffer (Invitrogen) and the
MyCycler, Thermal Cycler (Bio-Rad, Laboratories, Inc., Hercules,
CA) Cycling conditions: 25uC 5 mins, 50uC 45 mins, 70uC
15 mins. RNase H (Invitrogen) was used to remove RNA template.
The PCR was performed using HotStarTaq Master Mix (Qiagen).
Cycling conditions: 95uC 10 mins, 256(94uC 30 sec, 60uC 30 sec,
72uC 30 sec), 72uC 10 mins. mRNA from CD142PBMCs was
used as negative control for ALDH1A expression. The RT step for
quantitative PCR (qPCR) was carried out using the SuperScript
Vilo cDNA synthesis kit (Invitrogen). 100 ng of RNA was used in
each reaction. Relative qPCR was performed using the SYBR
Green PCR Master Mix (Applied Biosystems, Life Technologies).
1 ml of cDNA was used in each reaction. The 7000 Sequence
Detection System cycler (Applied Biosystems, Life Technologies)
was used for carrying out the reaction. Cycling conditions: 95uC
10 mins, 406(95uC 15 sec, 60uC 1 min). Dissociation curves were
generated to verify absence of unspecific amplification. Data were
analyzed using the ABI Prism 7000 SDS Software (Applied
Biosystems). The standard curve was generated using 2 fold
dilutions of RNA extracted from HSV-2 infected moDCs. RT of
standards was performed each time together with the samples to
avoid variation in the RT efficiency. In the real-time experiments
GAPDH was used as endogenous control for sample normalization
. Arbitraryunits were used to determinefoldincrease compared
to uninfected (mock) moDCs.
Analysis of ALDH activity
ALDH activity in individual cells was estimated using ALDE-
FLUOR staining kits (StemCell
BritishColumbia, Canada), according to the manufacturer’s
protocol with modifications as previously described . Briefly,
cells were suspended at 106cells/ml in ALDEFLUOR assay buffer
containing activated ALDEFLUOR substrate (150 nM) with or
without the ALDH inhibitor diethylaminobenzaldehyde (DEAB)
on ice. Cells were incubated for 45 mins at 37uC, washed and
stained with LIVE/DEAD fixable aqua (Invitrogen) for 30 mins
on ice in ALDEFLUOR assay buffer. They were washed again
and stained with APC anti-HLA-DR mAbs or isotype control for
30 mins on ice. ALDEFLUOR reactive cells were detected using
BD LSRII flow cytometer with 488-nm blue laser and standard
FITC 530/30 nm bandpass filter.
Cytokine and Chemokine analysis
Stored cell culture supernatants were thawed and examined for
CK/CC concentrations using the human cytokine 25-plex
(Invitrogen) on the Luminex 200 (Luminex Corp. Austin, Texas).
The kit measures IL-1b, IL-1RA, IL-2, IL-2R, IL-4, IL-5, IL-6,
IL-7, CXCL8, IL-10, IL-12p40, IL-13, IL-15, IL-17, IFNa, IFNc,
TNFa, GM-CSF, CCL2, CCL3, CCL4, CXCL9, CCL11, CCL5.
The StartStation software was used to analyze the data.
Animals HSV-2 challenge
Animals were challenged intra-rectally with 26108pfu of
replication competent or UV-inactivated HSV-2 in 1 ml of
serum-free RPMI 1640. Rectal swabs were collected 4 days and
1 day before HSV-2 challenge and 3 days and 6 days post
challenge. Swabs were drained of fluid and then discarded. The
remaining samples were centrifuged at 3500 rpm for 10 mins.
Total fluids or aliquots of supernatants were stored at 280uC.
Serum for SHIV-RT RNA levels was collected prior to challenge
and at time of necropsy. Plasma was separated from whole blood
by centrifugation at 2000 rpm for 10 mins, clarified at 2000 rpm
for 10 mins and stored at 280uC in 1 ml aliquots. SIV gag RNA
was measured by quantitative RT-PCR assay .
HSV-2 nested PCR
HSV-2 DNA shedding was determined by measuring the
presence of the HSV-2 gD gene (which encodes the viral entry
receptor glycoprotein D) using a nested PCR. gD primers used:
Ext FW AAGCGTGTTTACCACATTCAGCCG, RV TGTG-
TGATCTCCGTCCAGTCGTTT, Nested: FW TACTACG-
CAGTGCTGGAACG, RV CGATGGTCAGGTTGTACGTG.
This assay was able to reproducibly detect HSV-2 gD DNA signals
from 0.5 infected cells (single replicates) or 0.0005 infected cells (at
least 2 positives in 6 replicates) , DNA was extracted from
0.3 ml aliquots of the total fluids using DNeasy Blood & Tissue kit
(Qiagen). GAPDH primers and cycling conditions were performed
as described above in the methods for the ALDH1A PCR.
Animal cell isolation and flow cytometry
PBMCs were isolated from EDTA blood using Ficoll-Hypaque
density gradient centrifugation. Axillary, inguinal, iliac and MLNs,
as well as rectal tissues were obtained at necropsy. Fat tissue was
removed from the LNs with a scalpel, LNs were cut in small pieces
and passed through a 70 mm nylon cell strainer (BD-Falcon,
Franklin Lakes, NJ). Cells were washed twice with RPMI and
HSV-2-Infected DCs Expand a4b7high CD4+ T Cells
PLoS Pathogens | www.plospathogens.org11 June 2011 | Volume 7 | Issue 6 | e1002109
resuspended in FACS staining buffer. After removal of fat tissue
and blood vessels, from the rectal mucosa was cut in small pieces
and incubated in HBSS without Ca2+and Mg2+with 200 mg/ml
gentamycin (Gibco, Life Sciences), 7.5 mg DTT (Sigma-Aldrich)
and a final concentration of 1.7 mM of EDTA for 45 mins at
room temperature on a shaking platform. HBSS was discarded
and the remaining tissue washed in HBSS with Ca2+and Mg2+.
Tissues were incubated in R10 with 1 mg/ml hyaluronidase
(Sigma-Aldrich) 0.5 mg/ml Collagenase II (Sigma-Aldrich) 1 mg/
ml DNAse I (Roche) for 4 h at 37uC or until tissue was completely
digested. The cell suspension was passed through a 70 mm nylon
cell strainer, cells were washed twice with PBS and resuspended in
FACS wash buffer. Cells were stained with PerCP-Cy5.5 anti-
CD4 (clone SP34-2), Pacific Blue anti-CD3 (clone L200),
Alexa700 anti-CD69 (Clone FN50), FITC anti-CD95 (clone
DX2), APC anti-CD28 (clone 28.2), PE anti-dimeric a4b7 (Act-
1 Clone) and PE-Cy7 anti-CCR7 (clone 3D12). At least 200000
events were acquired in the lymphocyte gate. Samples were
analyzed using the BD LSRII Flow Cytometer.
Wilcoxon signed-rank and Mann-Whitney non-parametric tests
were used to compare variables between groups (mock versus live
HSV-2, UV-HSV-2 versus live HSV-2 and mock versus UV-
HSV-2). Wilcoxon signed-rank and one sample t test with 1 as
hypothetical value were both used to analyze results expressed as
fold increase. A two-tailed P value a,0.05 was considered
significant. Analysis was performed using Prism (GraphPad
Software, Inc) version 5a.
NCBI Reference Sequences:
NM_170696.1 mRNA ALDH1A3: NM_000693.2
Swiss-Prot: ICP-8 P11870.2
NM_000689.3 mRNA ALDH1A2:
Gating strategy for a4b7+CD4+T cells in rectal
mucosa. Cells isolated from rectal mucosa of clinically stable
SHIV-RT-infected macaques were stained with the LIVE/DEAD
discriminator fixable Aqua and mAbs against CD3, CD4, CD95
and a4b7. The respective gates are indicated in each panel.
are viable 24 h post infection. A) moDCs were infected with
0.04, 0.2, 1 MOI of HSV-2 or treated with HSV-2 growing
media (MOCK) and 24 h later stained with Annexin/PI. The
percentages of early apoptotic cells (Annexin V+PI2) and late
apoptotic cells (Annexin V+
independent experiments. B) 24 h HSV-2-infected (0.2 MOI),
UV-HSV-2 or mock-treated moDCs were stained with the
LIVE/DEAD BD fixable Aqua dye. The percentages of
Aqua-low viable cells for 1 of at least 5 independent experiments
Phenotype of the a4b7highCD4+T cells in the
DC-T cell co-cultures. CD4+T cells were co-cultured with
HSV-2-infected DCs for 5 days. a4b7highT cells are CD45RO+
(left). a4b7highCD45RO+T cells are CD62L+CCR7+(right). Plots
are representative of more than 15 independent experiments.
moDCs infected with 0.2 MOI of live HSV-2
PI+) are shown for 1 of 2
do not induce a4b7 up-regulation on T cells. Mock-,
UV-HSV-2- (0.2 MOI and 5 MOI), or Live HSV-2- (0.2 MOI)
treated DCs were mixed with autologous CD4+T cells and
cultured for 5 days. The fold changes (mean 6 SEM, n=11 for
the mock and 0.2 MOI of UV or live HSV-2 conditions and n=3
with 5 MOI of UV-HSV-2 condition) in the percentage of
a4b7highCD3+CD4+T cells (A) and of CD69+CD3+CD4+T cells
(B) are shown. (*p,0.05, **,0.01, ***p#0.001).
Exposure of CD4+T cells to live HSV-2 does
not induce up-regulation of a4b7. A-C) CD4+T cells were
exposed to 0.2 and 5 MOI of live HSV-2, UV-HSV-2 (0.2 MOI)
or HSV-2 growing media (MOCK) for 5 days. The fold changes
(mean 6 SEM; 4 independent experiments) in the percentage of
ICP-8+(A), of a4b7+in the low, intermediate and high subsets (B)
and of CD69+(C) cells are shown for each treatment group. In
panels B and C, there was no difference in the percentages of
a4b7+and CD69+cells in cultures exposed to 0.2 versus 5 MOI
and so the data have been combined (B–C; total of 8 independent
moDCs treated with a high dose of UV-HSV-2
effect on ALDH1A1 expression and HIV-1 replication. A)
The fold changes (mean 6 SEM, n=13 for the mock, 0.2 MOI of
UV and Live HSV-2 conditions and n=6 for the 5 MOI of UV-
HSV-2 condition) in ALDH1A1 mRNA of LIVE and UV-HSV-2
treated moDCs (24 h post treatment) are shown relative to mock
moDCs (set as 1). B) The fold changes (mean 6 SEM, n=15 the
mock, 0.2 MOI of UV and Live HSV-2 conditions and n=8 for
the 5 MOI of UV-HSV-2 condition) in HIV-1 DNA copies/cell
after 5 days of co-culture are shown relative to the MOCK
controls (set as 1). (*p,0.05, **,0.01, ***p#0.001).
Exposure of CD4+T cells to live HSV-2 does
not significantly increase HIV-1 replication. The fold
changes (mean 6 SEM, 10 independent experiments) in HIV-1
copies/cell in CD4+T cells co-exposed to HIV-1 and 0.2 or 5
MOI of live, UV-HSV-2 (0.2 MOI) or HSV-2 growing media
(MOCK) for 5 days. (B) The fold changes (mean 6 SEM, 4
independent experiments) in the HIV-1 copies/cell in presence (+)
or absence (2) of the RARa antagonist are shown relative to the
mock-treated controls (set as 1).
moDCs treated with high dose UV-HSV-2:
HSV-2 status. CD4 counts before (BL) and after (D6 p.i.) HSV-2
challenge. SHIV-gag RNA levels in serum before (BL) and after
(D6 p.i.) HSV-2 challenge. HSV-2 DNA detected (+) or not (2)
before (BL) and 3 and 6 days (D3 and D6 p.i.) after HSV-2
challenge (LIVE) or treatment with UV inactivated HSV-2 (UV).
Summary of CD4 counts, SIV viral loads and
ALDH1A Primers. Primer sequences used for RT-
Conceived and designed the experiments: EM MR. Performed the
experiments: EM HT IF. Analyzed the data: EM MR. Contributed
reagents/materials/analysis tools: MPJ JDL JB AG JA. Wrote the paper:
EM MR. Contributed to the discussion: JA.
HSV-2-Infected DCs Expand a4b7high CD4+ T Cells
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