Differential susceptibility of naïve, central memory and effector memory T cells to dendritic cell-mediated HIV-1 transmission.

ral
ssBioMed CentRetrovirology
Open AcceResearch
Differential susceptibility of naïve, central memory and effector
memory T cells to dendritic cell-mediated HIV-1 transmission
Fedde Groot1,2, Toni MM van Capel2, Joost HN Schuitemaker2,
Ben Berkhout1 and Esther C de Jong*2
Address: 1Dept. of Human Retrovirology, Academic Medical Centre, University of Amsterdam, Amsterdam, The Netherlands and 2Dept. of Cell
Biology and Histology, Academic Medical Centre, University of Amsterdam, Amsterdam, The Netherlands
Email: Fedde Groot - info@feddegroot.nl; Toni MM van Capel - t.m.vancapel@amc.uva.nl;
Joost HN Schuitemaker - j.schuitemaker@iqcorporation.nl; Ben Berkhout - b.berkhout@amc.uva.nl; Esther C de Jong* - e.c.dejong@amc.uva.nl
* Corresponding author
Abstract
Background: Dendritic cells (DC) have been proposed to facilitate sexual transmission of HIV-1
by capture of the virus in the mucosa and subsequent transmission to CD4+ T cells. Several T cell
subsets can be identified in humans: naïve T cells (TN) that initiate an immune response to new
antigens, and memory T cells that respond to previously encountered pathogens. The memory T
cell pool comprises central memory (TCM) and effector memory cells (TEM), which are
characterized by distinct homing and effector functions. The TEM cell subset, which can be further
divided into effector Th1 and Th2 cells, has been shown to be the prime target for viral replication
after HIV-1 infection, and is abundantly present in mucosal tissues.
Results: We determined the susceptibility of TN, TCM and TEM cells to DC-mediated HIV-1
transmission and found that co-receptor expression on the respective T cell subsets is a decisive
factor for transmission. Accordingly, CCR5-using (R5) HIV-1 was most efficiently transmitted to
TEM cells, and CXCR4-using (X4) HIV-1 was preferentially transmitted to TN cells.
Conclusion: The highly efficient R5 transfer to TEM cells suggests that mucosal T cells are an
important target for DC-mediated transmission. This may contribute to the initial burst of virus
replication that is observed in these cells. TN cells, which are the prime target for DC-mediated X4
virus transmission in our study, are considered to inefficiently support HIV-1 replication. Our
results thus indicate that DC may play a decisive role in the susceptibility of TN cells to X4 tropic
HIV-1.
Background
Several CD4+ T cell subsets can be identified in humans:
naïve T cells (TN) to mount an immune response to a vari-
ety of new antigens, and memory T cells to respond to pre-
using high endothelial venules to enter lymph nodes [1].
The memory T cell pool comprises distinct populations of
central memory (TCM) and effector memory T cells (TEM),
characterized by distinct homing and effector function
Published: 17 August 2006
Retrovirology 2006, 3:52 doi:10.1186/1742-4690-3-52
Received: 21 June 2006
Accepted: 17 August 2006
This article is available from: http://www.retrovirology.com/content/3/1/52
© 2006 Groot 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.Page 1 of 10
(page number not for citation purposes)
viously encountered pathogens. TN cells preferentially
circulate between blood and secondary lymphoid tissues,
[2,3]. Like TN cells, TCM cells express CCR7 and CD62L,
two receptors required for migration to T cell areas of sec-

Retrovirology 2006, 3:52 http://www.retrovirology.com/content/3/1/52
ondary lymphoid tissue. They furthermore have limited
effector function, but can proliferate and become TEM cells
upon secondary stimulation with antigen, and therefore
play a role in long term protection. TEM cells have lost
CCR7 expression, and home to peripheral tissues and sites
of inflammation to provide immediate protection against
pathogens [2,3]. Consequently, TN and TCM cells are pri-
marily found in blood and lymphoid tissue, whereas TEM
cells are enriched in gut, liver and lung. Within the TEM cell
subset, effector Th1 and Th2 cells are recognized, which
are classified by different functional properties based on
unique cytokine profiles. Th1 cells produce high levels of
IFNγ and TNFβ, which is instrumental in cell-mediated
immunity against intracellular pathogens like viruses. Th2
cells secrete a large variety of cytokines (IL-4, IL-5, IL-9
and IL-13) that are crucial for the clearance of parasites,
like helminths. Both types of effector cells play a role in
the induction of a humoral (antibody) response against
different extracellular pathogens [4].
Sexual transmission of HIV-1 involves the crossing of
mucosal tissue by the virus, and several studies have
shown that one of the very first cell types encountered are
intraepithelial and submucosal dendritic cells (DC). Con-
sequently, they have been proposed to facilitate HIV-1
transmission and infection [5-8]. DC are professional
antigen presenting cells that sample the environment at
sites of pathogen entry. Sentinel immature DC (iDC)
develop into mature effector DC (mDC) upon activation
by microorganisms or inflammatory signals, and migrate
to the draining lymph nodes where they encounter and
stimulate naïve Th cells [9,10]. DC are able to capture
HIV-1 by a range of receptors, of which the best studied
example is DC-SIGN [11]. Subsequent transmission to T
cells takes place in lymph nodes via cell-cell contact
through an 'infectious synapse' [12]. Additionally, DC can
support local virus replication in T cells present in the
mucosal tissue [7,8].
An increasing number of studies on HIV-1 and SIV dem-
onstrate that the initial burst of viral replication takes
place in CCR5+ CD4+ (effector) memory T cells in the lam-
ina propria of mucosal tissues [13-18]. CCR5 and CXCR4
are the major co-receptors used by HIV-1, with CCR5
being the initial co-receptor used by the virus after trans-
mission. This receptor is primarily expressed on the mem-
ory T cell subset and macrophages [19]. Over time, HIV-1
starts to use CXCR4 in some patients, thereby expanding
its target cell repertoire to TN cells, coinciding with faster
disease progression [20,21].
Because DC play an important role in HIV-1 pathogene-
sis, and TN, TCM and TEM cells have distinct functions and
that CCR5-using (R5) HIV-1 is efficiently transmitted to
TEM cells but not to TN cells. Transmission to TCM cells was
of intermediate efficiency. Transmission to pure popula-
tions of Th1 or Th2 cells, or to an unbiased population
containing both types (Th0) was equally efficient. The
highly efficient R5 transfer to TEM cells suggests that
mucosal (TEM) cells are an important target for DC-medi-
ated transmission, which may contribute to the observed
initial burst of virus replication in these cells. CXCR4-
using (X4) HIV-1 could be transmitted to all T cell subsets,
due to expression of CXCR4 on all subsets. Surprisingly,
X4 HIV-1 was preferentially transmitted to TN cells, which
are considered to inefficiently replicate X4 HIV-1 [22-24].
This study shows that co-receptor expression is a decisive
factor for DC-mediated HIV-1 transmission, and more
importantly, that DC may play a crucial role in making TN
cells susceptible to X4 HIV-1 replication later in infection.
Results
T cell subsets differ in susceptibility to DC-mediated
transmission of R5 and X4 HIV-1
To investigate whether different CD4+ T cell subsets differ
in their susceptibility to DC-mediated HIV-1 transmis-
sion, we isolated by live sorting highly purified popula-
tions of CD45RA+ CD45RO- naïve T cells (TN) and
CD45RA- CD45RO+ memory T cells from pure CD4+ T
cells. Based on the expression of CCR7, a homing receptor
for secondary lymphoid tissue, the memory pool was fur-
ther divided in CCR7+ central memory T cells (TCM) and
CCR7- effector memory T cells (TEM) [2,3]. We subse-
quently incubated DC with the R5 virus JR-CSF isolate or
the X4 virus LAI isolate for 2 hr, followed by washing steps
to remove unbound virus. After addition of the respective
T cell subsets, we determined the transmission efficiency
by measuring the accumulation of HIV-1 capsid protein
p24 (CA-p24) in T cells by FACS. To prevent subsequent
rounds of HIV-1 replication after transmission in this sin-
gle-cycle transmission assay, we added an inhibitor of the
viral protease (saquinavir, [25,26]).
In a control experiment without HIV-1, no CA-p24 posi-
tive CD3+ T cells were scored (Fig. 1A). Addition of R5
HIV-1 resulted in high percentages of CA-p24+ TEM cells,
and hardly any CA-p24+ TN cells (2.9 and 0.1 %, respec-
tively). The transmission to TCM cells was of intermediate
efficiency (1.9%). With X4 HIV-1, the pattern was
reversed: X4 HIV-1 was preferentially transmitted to TN
cells (4%), then to TCM cells (2.2%), and the transmission
to TEM cells was least efficient (1.4%) (Fig. 1A). Overall, X4
transmission was more efficient than R5 transmission,
and could take place to all subsets. For both viruses, the
percentage CA-p24+ T cells reached a maximum value 2
days post transmission, and these data are quantified inPage 2 of 10
(page number not for citation purposes)
locations in the body, we set out to investigate the contri-
bution of DC in infection of these T cell subsets. We found
Fig. 1B. This experiment demonstrates that there is not
one exclusive T cell subset that is the preferred target of

Retrovirology 2006, 3:52 http://www.retrovirology.com/content/3/1/52
DC-mediated HIV-1 transmission, but that the efficiency
depends on the tropism of the transmitted virus.
DC-mediated HIV-1 transmission is co-receptor dependent
The different transmission patterns for R5 and X4 HIV-1
prompted us to investigate the co-receptor expression on
each T cell subset (Fig. 2A). We found that the level of co-
receptor expression for both CCR5 and CXCR4 correlates
with the transmission efficiencies depicted in Fig. 1B:
sets, but its expression declines from TN cells via TCM to
TEM cells.
To investigate the role of co-receptor expression in DC-
mediated HIV-1 transmission, we added the well-
described inhibitors RANTES and AMD3100 in the single-
cycle transmission assay. These compounds inhibit HIV-1
infection of T cells by blocking the co-receptors CCR5 and
DC-mediated HIV-1 transmission is co-receptor dependentFigur 2
DC-mediated HIV-1 transmission is co-receptor
dependent. (A) FACS analysis of TN, TCM and TEM cells for
CD4 and co-receptors CCR5 and CXCR4. Open histograms
represent isotype controls. (B) Transmission inhibition by
co-receptor ligands and a fusion inhibitor. A single-cycle
transmission assay to TN, TCM and TEM cells was performed
with R5 and X4 HIV-1 loaded DC. Prior to co-culture with
DC, the T cells were pre-incubated with ligands for CCR5
(RANTES) or CXCR4 (AMD3100) (grey bars) or alterna-
tively, with fusion inhibitor T1249 (black bars). After 2 days,
the percentage CA-p24+ T cells was determined by FACS.
The percentage inhibition of transmission relative to trans-
mission without inhibitors is indicated on the y-axis. Error
bars represent standard deviations.
A
B
CD4 CCR5 CXCR4
0
20
40
60
80
100
120
140
%
in
hi
bi
tio
n
co-receptor block (RANTES or AMD3100)
fusion inhibitor (T1249)
TN
TCM
TEM
TN TCM TEM
R5 HIV-1
TN TCM TEM
X4 HIV-1
T cell subsets differ in susceptibility to DC-mediated trans-mission of R5 and X4 HIV-1Figure 1
T cell subsets differ in susceptibility to DC-mediated
transmission of R5 and X4 HIV-1. (A) DC were incu-
bated with R5 or X4 HIV-1, or mock treated, followed by
extensive washing to remove unbound virus. DC were sub-
sequently co-cultured with CD4+ naïve T cells (TN), central
memory T cells (TCM) or effector memory T cells (TEM) in
the presence of saquinavir to prevent spreading infection
(single-cycle transmission assay). Two days after transmis-
sion, T cells were harvested and stained for CD3 and intrac-
ellular CA-p24 to determine the percentage HIV+ T cells.
Representative FACS plots are shown. (B) Summary of one
representative experiment. Error bars represent standard
deviations. * p < 0.05 ; ** p < 0.01; *** p < 0.001.
no virus R5 HIV-1 X4 HIV-1
TN
TCM
TEM
CD3 CD3CD3
CD3 CD3CD3
CD3 CD3CD3
C
A-
p2
4
C
A-
p2
4
C
A-
p2
4
C
A-
p2
4
C
A-
p2
4
C
A-
p2
4
C
A-
p2
4
C
A-
p2
4
C
A-
p2
4
A
B
0
1
2
3
4
5
TN TCM TEM
R5 HIV-1
%
C
A-
p2
4+
T
c
el
ls
TN TCM TEM
X4 HIV-1
***
*
**
*
*** *Page 3 of 10
(page number not for citation purposes)
CCR5 expression is most pronounced on TEM cells, and is
undetectable on TN cells; CXCR4 is detectable on all sub-
CXCR4, respectively [27,28]. Transmission of HIV-1 was
completely blocked through the addition of these com-

Retrovirology 2006, 3:52 http://www.retrovirology.com/content/3/1/52
pounds (Fig. 2B, grey bars). We furthermore could block
transmission completely with inhibitor T1249 (Fig. 2B,
black bars). This peptide prevents fusion of viral and cel-
lular membranes [29]. Our results thus demonstrate that
DC-mediated HIV-1 transmission requires 'regular' infec-
tion through CD4 and a co-receptor.
Method of T cell stimulation determines HIV-1
susceptibility
In addition to quantification of the transmission effi-
ciency in a single-cycle transmission assay (Fig. 1 and 2),
we followed viral replication after transmission (Fig. 3). In
this spreading infection assay, we did not add saquinavir
to allow cell-cell spread of newly produced virus. Replica-
tion of R5 and X4 HIV-1 in TN, TCM and TEM cells following
DC-mediated transmission reflects the results of the sin-
gle-cycle transmission assay: R5 HIV-1 preferentially rep-
licates in memory T cells, whereas X4 HIV-1 prefers TN
cells over the memory subsets (Fig. 3A and 3B).
Since this spreading infection assay involves two different
steps, i.e. transmission and subsequent replication, we
also studied R5 and X4 HIV-1 replication in TN, TCM and
TEM cells in a DC-independent system. Therefore, cellular
Spreading infection assayFigure 3
Spreading infection assay. Replication of R5 (A) and X4 (B) virus in TN, TCM and TEM cells after DC-mediated HIV-1 trans-
mission. Alternatively, the T cell subsets were stimulated by crosslinking CD3/CD28 with antibodies and infected with R5 (C)
D
A
0
100
200
300
400
500
600
700
0 2 4 6 8 10 12
days post transmission
C
A-
p2
4
(n
g/
m
l)
TNTCMTEM
R5 HIV-1
C
0
10
20
30
40
50
60
70
80
90
0 2 4 6 8 10 12
days post infection
C
A-
p2
4
(n
g/
m
l)
TNTCMTEM
R5 HIV-1
B
0
50
100
150
200
250
300
350
400
0 2 4 6 8 10 12
days post transmission
C
A-
p2
4
(n
g/
m
l)
TNTCMTEM
X4 HIV-1
0
50
100
150
200
250
300
350
400
450
0 2 4 6 8 10 12
days post infection
C
A-
p2
4
(n
g/
m
l)
TNTCMTEM
X4 HIV-1Page 4 of 10
(page number not for citation purposes)
or X4 (D) virus. Viral replication was followed by CA-p24 ELISA on the supernatant. Error bars represent standard deviations.

Retrovirology 2006, 3:52 http://www.retrovirology.com/content/3/1/52
proliferation was induced by cross linking of CD3 and
CD28 on the T cells with antibodies (Fig. 3C and 3D). As
expected, the susceptibility of all T cell subsets to R5 HIV-
1 replication was low after CD3/CD28 stimulation. This
phenomenon was previously described for CD4+ T cells in
general, and is the consequence of CCR5 down regulation
and production of natural CCR5 ligands that compete for
co-receptor binding [30,31]. But despite this low replica-
tion capacity, the pattern of R5 replication was compara-
ble to the replication after DC-mediated transmission of
R5 HIV-1: replication was lower in TN cells. Surprisingly,
X4 replication in TN cells was significantly delayed in com-
parison to TCM and TEM cells, which does not reflect the
enhanced transmission and replication in TN cells in the
transmission experiments (Fig. 1 and 3B).
This discrepancy prompted us to compare HIV-1 replica-
tion in T cells stimulated by either DC or α-CD3/CD28
antibodies, without any complicating factors like trans-
mission steps. We therefore stimulated all T cell subsets
with DC, or alternatively, with α-CD3/CD28 antibodies
and harvested the T cells after 4 days of proliferation. The
cells were subsequently infected with X4 HIV-1. DC-stim-
ulated TN cells were more susceptible to X4 HIV-1 replica-
tion than the memory subsets (Fig. 4A), which reflects the
replication after transmission (Fig. 3B). The reverse was
observed with α-CD3/CD28 stimulated T cells (Fig. 4A),
which is in concordance with the results of Fig. 3D in
which the cells were infected immediately after stimula-
tion. This indicates that the enhanced replication of X4
HIV-1 in TN cells following DC-mediated transmission, is
due to a higher HIV-1 susceptibility. It further demon-
strates that crosslinking of CD3 and CD28 by antibodies
is not comparable to DC-T cell stimulation, although this
crosslinking is considered to mimic DC encounter. The
difference between both stimulation methods is further
manifested by the proliferative capacity of the T cells, as
determined by 3H-thymidine incorporation (Fig 4B). The
proliferation pattern of the different T cell subsets after
DC or α-CD3/CD28 stimulation is clearly not the same.
DC transmit HIV-1 with equal efficiency to Th1 and Th2
cells, or to an unpolarized population
The TEM cell subset can be further divided into effector Th1
and Th2 cells [4]. We generated in vitro polarized popula-
tions of pure Th1 and Th2 cells, or an unbiased popula-
tion containing both types (Th0 cells), by culturing
purified TN cells with or without IL-12 or IL-4, as previ-
ously described [32]. We next investigated whether HIV-1
is differently transmitted to these subsets of effector Th1,
Th2 or Th0 cells. In addition, we tested different mature
DC subsets. Depending on the type of pathogen and tis-
sue factors, immature DC develop into mature effector
Th2 cells, designated DC1 and DC2 respectively [33].
DC0 induce an unpolarized response (Th0). DC0, DC1
and DC2 were generated by culturing immature DC with
maturation factors (MF, IL-1β and TNFα) only (DC0), or
MF with either IFNγ (DC1) or prostaglandin E2 (DC2)
[34].
The intracellular cytokine profiles of the effector Th cell
populations were analyzed by FACS (Fig 5A). The Th1
population consists primarily of IFNγ producers, whereas
the Th2 population contains mostly IL-4 producers. The
unpolarized Th0 population is composed of both cell
types. All T cell subsets expressed similar levels of CCR5
Method of T cell stimulation determines HIV-1 susceptibilityFigure 4
Method of T cell stimulation determines HIV-1 sus-
ceptibility. (A) Comparison of viral replication in TN, TCM
and TEM cells that were stimulated by DC or by CD3/CD28
crosslinking with antibodies. The T cells were stimulated for
4 days, harvested and re-plated before infection with X4
HIV-1. Viral spread was followed by CA-p24 ELISA, of which
the results of day 6 are shown. (B) To measure T cell prolif-
eration TN, TCM or TEM cells were incubated with DC or α-
CD3/CD28 antibodies and after 4 days, cellular proliferation
was determined by 3H-thymidine incorporation. Error bars
represent standard deviations. * p < 0.05 ; ** p < 0.01; *** p
< 0.001.
A
B
0
100
200
300
400
500
600
700
DC α-CD3/28
C
A-
p2
4
(n
g/
m
l)
0
10000
20000
30000
40000
DC α-CD3/28
3H
-T
dR
(c
pm
)
TN
TCM
TEM
TN
TCM
TEM**
*** ***
*
*
**
*
***
*Page 5 of 10
(page number not for citation purposes)
DC that are specialized to stimulate naïve T cells to
develop into IFNγ-producing Th1 cells or IL-4-producing
and CXCR4, and proliferated to a comparable extent, as
determined by 3H incorporation (results not shown).