http://intimm.oxfordjournals.org/content/early/2004/10/04/intimm.dxh164.full.pdf

International Immunology, Vol. 16, No. 11, pp. 1623–1631
doi:10.1093/intimm/dxh164 Advance Access publication 4 October 2004
ª 2004 The Japanese Society for Immunology
Recall antigen activation induces prompt release of
CCR5 ligands from PBMC: implication in memory
responses and immunization
Lingling Sun, Sayed F. Abdelwahab1, George K. Lewis and Alfredo Garzino-Demo
Institute of Human Virology, University of Maryland Biotechnology Institute, 725 West Lombard Street, Room S613,
Baltimore, MD 21201-1192, USA
1
Present address: Department of Microbiology and Immunology, El-minia Faculty of Medicine, El-minia University,
El-minia 61111, Egypt
Keywords: chemokines, lymphoproliferation, MIP-1a, MIP-1b, RANTES
Abstract
CCR5 ligands RANTES, macrophage inflammatory protein (MIP)-1a and MIP-1b are potent and specific
inhibitors of strains of human immunodeficiency virus (HIV) that use CCR5 as a receptor, which are
the strains most involved in primary infection. Recently, we observed that release of CCR5 ligands is
a consistent and reproducible parameter of response to antigen activation in studies using PBMC. In this
study, we show that CCR5 ligands are released upon antigen [Fragment C of tetanus toxin (TTC)]
stimulation in 81% (n 5 16) of subjects tested, as detected by a standard ELISA in tissue culture
supernatants of antigen-activated cells. In contrast, ELISA for other cytokines from the same
supernatants revealed that IFN-c release could be detected only in 31% of subjects, IL-2 could be
detected only in 12% of the subjects and IL-4 was not detectable in any of the subjects tested. Similarly,
proliferative responses to TTC, as measured by a standard tritiated thymidine incorporation assay, were
detectable in only 56% of the subjects. Similar observations have been reported in flow cytometric
studies, and resonate with previous findings emphasizing the role of CCR5 in T cell responses. In
addition, the levels of CCR5 ligands in supernatants from antigen-activated cells were sufficient to
inhibit infection of R5 HIV. Thus, CCR5 ligands might play a role in controlling HIV in vivo. Taken
together, these observations suggest that CCR5 ligands, and in particular MIP-1a and MIP-1b, released in
the course of memory responses may play a role in protecting CD41 memory T cells from infection.
Introduction
Several surrogate markers have been studied to investigate
memory T cell responses. Traditionally, since response to antigenic stimulation triggers a clonal expansion of antigen-specific
cells, lymphoproliferative responses have been evaluated, by
using assays such as tritiated thymidine ([3H]TdR) uptake.
Immune responses are also known to trigger the release of
several cytokines, which have also been used as surrogate
markers. More recently, the role of chemokines in the elicitation of immune responses has been more thoroughly evaluated
[for reviews on this subject, see refs (1, 2)]. Specific expression
of chemokine receptors allows for migration of select cell
subsets to sites of antigen presentation. Therefore, while some
degree of redundancy exists in the chemokine system, it is
possible to ascribe involvement of certain chemokines with
components of the immune response (1, 2). For example,
CCR5, which is expressed in monocytes, immature dendritic
cells and Th1 cells, has been involved in memory responses (1,
2). The ligands for CCR5 are the chemokines RANTES,
macrophage inflammatory protein (MIP)-1a and MIP-1b.
Interestingly, we had demonstrated that these chemokines
are potent and specific inhibitors of certain isolates of human
immunodeficiency virus (HIV)-1, i.e. those that infect macrophages and primary cells (3). Subsequently, it has been shown
that this interference is due to the fact that macrophage-tropic
isolates of HIV enter cells through CCR5 (4–8), hence their
current definition as R5 tropic, as opposed to X4, i.e. using
CXCR4 (9), the receptor for the chemokine stromal cell derived
factor (SDF-1), or R5X4, for isolates that can use both receptors. Consequently, CCR5 has a dual role, as a receptor for
chemokines and as a docking site for HIV infection. Therefore,
Correspondence to: A. Garzino-Demo; E-mail: [email protected]
Transmitting editor: G. Trinchieri
Received 10 March 2004, accepted 26 August 2004
1624 CCR5 ligands release in immune memory responses
we became interested in studying the mechanism of CCR5
ligands release in immune response, hypothesizing that their
expression in memory responses might provide a novel
immunological marker that may participate in controlling HIV
infection. To this end, we studied chemokine release in an HIV
cohort, stimulating cells from seropositive and seronegative
subjects, and from control subjects not enrolled in the cohort.
Our study showed that acquired immunodeficiency syndrome
(AIDS)-free status and higher CD4+ T cell counts correlate with
higher CCR5 ligands release from PBMC after in vitro
activation by HIV antigens (10). These studies are in
agreement with published reports from other groups: Furci
et al. (11) showed that CD4+ T cells from HIV+ individuals
release RANTES, MIP-1a and MIP-1b at levels sufficient to
block HIV infection when stimulated with HIV antigens. Ferbas
et al. (12) observed a similar antigen-specific response in
D32CCR5 heterozygous individuals, whose viral load is
inversely correlated with antigen-stimulated chemokine production. While CCR5 ligands are also released from CD8+
T cells in an antigen-specific manner (13, 14), several studies
have shown that CD4+ T cells secrete them at high levels (15–
19): besides the studies on antigen activation and release of
CCR5 ligands quoted above (10–12), it has been shown that
CD4+ T cells from exposed uninfected individuals release
CCR5 ligands (15, 16), and that CD4+ T cell clones from longterm non-progressing HIV+ subjects release high levels of
CCR5 ligands (17, 18). In addition, a study had shown that
CCR5 ligands are part of the vigorous, HIV-1-specific CD4+
response associated with control of HIV viremia (19). Interestingly, a recent study has shown that, despite the finding
that HIV-specific CD4+ memory T cells are the main targets for
HIV infection, a significant number of memory cells do not get
infected by HIV, even at high multiplicity of infection (20). There
is no evident explanation for this discrepancy, which may be
related to the release of anti-viral factors, such as CCR5
ligands, from activated cells. Thus, we wanted to investigate
whether the frequency of cognate cells to a recall antigen is
high enough to induce levels of CCR5 ligands sufficient to
inhibit HIV replication, and whether the accumulation of
suppressive levels of CCR5 ligands occurs in a time frame
compatible with fusion/entry events [i.e. within 2 h of virus/cell
or cell/cell contact (21, 22)]. To this end, we evaluated the
release of CCR5 ligands from PBMC in response to a classic
recall antigen, Fragment C of tetanus toxin (TTC). We observed
that CCR5 ligands are promptly released upon TTC activation,
and that their release, as measured by ELISA, constitutes
a marker of memory response more sensitive than IFN-c, IL-2
or IL-4 release, or than lymphoproliferation (as measured by
thymidine incorporation). This prompt release appears to be
due to pre-existing accumulation of CCR5 ligands. Finally,
levels of CCR5 ligands released upon antigenic stimulation are
sufficient to inhibit replication of R5 HIV, suggesting that
antigen-stimulated cells can contribute to protection against
HIV infection.
manufacturer (Sigma Chemical Co., St Louis, MO, USA). All
the donors signed informed consent forms approved by the
Institutional Review Board. The lymphocyte proliferation assay
was modified from ACTG protocol 209 (10). At each time point,
fresh PBMC were cultured in round-bottom 96-well plates
(Falcon-BD, Franklin Lakes, NJ, USA) in RPMI medium
(GIBCO–Invitrogen, Carlsbad, CA, USA) supplemented with
10% human AB serum and antibiotics (100 U ml1 penicillin,
100 mg ml1 streptomycin) (GIBCO–Invitrogen) at a density of
2 3 105 cells per well. Cells were incubated with media alone
or with 3–20 lg ml1 of TTC (Calbiochem, La Jolla, CA, USA),
10 mg ml1 Candida albicans (Greer Laboratories, Lenoir, NC,
USA) or 2.5 lg ml1 PHA (Sigma). All lymphocyte proliferation
assays were done in triplicate. After incubation at 37C, 5%
CO2 (duration of incubation varied, see Results), the cells were
pulsed with 1 lCi per well of [3H]TdR in complete RPMI. Cells
were harvested and incorporated [3H]TdR was determined
by scintillation counting. Geometric mean counts per minute
(c.p.m.) were calculated for the triplicate wells with and
without antigen. Results were expressed as raw data or as
‘lymphocyte stimulation index’, which is the geometric mean
c.p.m. of the cells plus antigen divided by the geometric mean
c.p.m. of the cells without antigen (medium alone). Supernatants were collected at the time points indicated in Results
and frozen at 70C for RANTES, MIP-1a and MIP-1b ELISAs.
All b-chemokine assays were performed by commercial ELISA
(R&D Systems, Minneapolis, MN, USA). In some experiments,
5 lg ml1 actinomycin D (Sigma), 2.5 lg ml1 cycloheximide
(Calbiochem) or 5 lg ml1 brefeldin A (BFA, Sigma) was
added to cell culture media for up to 6 h.
Flow cytometry
Methods
Whole-blood samples collected from healthy donors were
activated with 10 lg ml1 of TTC (Calbiochem) or Staphylococcus aureus enterotoxin B (SEB; Sigma) at 37C for 1 h as
previously described (23). Then, the blood sample was treated
with BFA (10 lg ml1, Sigma) for an additional 5 h. After 6 h,
100 ll of 20 mM EDTA (for a final concentration of 2 mM EDTA)
was added directly to the whole-blood cultures. Blood samples
were lysed and fixed in 14 ml of 13 FACS lysing solution (BD,
San Jose, CA, USA) for 10 min at room temperature (RT). Cells
were washed in wash buffer, PBS with 2% heat-inactivated
human serum from AB donors and 0.1% sodium azide (Sigma),
and subsequently re-suspended in 0.5 ml of FACS permeabilizing solution (BD) for 10 min at RT. After permeabilization,
cells were washed once again and stained with fluoresceinconjugated antibodies specific for MIP-1b (R&D Systems),
CD3 [BD; labeled with peridinin-chlorophyll protein (PerCP)],
CD69 (BD; labeled with PE), IFN-c (BD; labeled with PE) or
CD4 [BD; labeled with allophycocyanin (APC)] for 30 min at RT
in the dark. Finally, labeled cells were washed two times with
wash buffer, fixed with 1% PFA (Fisher, Fair Lawn, NJ, USA)
and stored at 4C. Four-color flow cytometric analysis was
preformed by using a FACSCalibur (Becton Dickinson, Franklin
Lakes, NJ, USA). Data analysis was carried out using the flowjo
software (Tree Star, San Carlos, CA, USA).
Cells and laboratory studies
Infectivity assay
Fresh PBMC were obtained by Hystopaque separation from
whole blood of healthy donors, following the instructions of the
PHA-activated PBMC (1 3 105 PBMC per well) were infected
for 2 h with 100 50% tissue culture infective dose (TCID50) of
CCR5 ligands release in immune memory responses 1625
HIVBaL stock of HIV-1BaL, an R5 isolate. After 2 h, cells were
washed three times with PBS and complete media added with
25 or 50% of supernatants from cells activated with TTC or
PHA. As controls, we used supernatants from cells that had
not been activated, and we infected PBMC without treatment
with supernatants from antigen activation. We tested supernatants obtained from cells from different donors and
harvested at 8 or 24 h after activation. In some cases, we
tested the supernatants in the presence or absence of
a cocktail of neutralizing polyclonal IgG (Nab) against CCR5
ligands [100 lg ml1 each of anti-RANTES, anti-MIP-1a and
anti-MIP-1b (R&D Systems)].
Statistical analysis
Analyses of data distribution and of statistical significance
using paired two-tailed t-tests were performed using GraphPad InStat, version 3.00, for Windows 95 (GraphPad Software,
San Diego, CA, USA).
Results
Antigen-induced chemokine release and proliferation
Our data obtained with antigen stimulation using HIV antigens
and C. albicans show that these antigens induce measurable
production of CCR5 chemokines (10). Thus, it appeared that
memory responses could trigger CCR5 ligands release. To
investigate this hypothesis, we measured chemokine release
response to a classical recall antigen, i.e. TTC. We obtained
freshly harvested PBMC from healthy volunteers vaccinated
against tetanus and cultured them in media without stimulation
or activated with TTC (at concentrations of 3, 10 and 20 lg
ml1). Positive controls included C. albicans and PHA at 10 lg
and 2.5 lg ml1, respectively. The cultures were incubated for
up to 9 days at 37C/5% CO2. At days 3, 6 and 9, an aliquot of
cells was used for a proliferation assay by [3H]TdR uptake,
while supernatants were used for RANTES, MIP-1a and
MIP-1b assays by ELISA. Our results show that as little as
3 lg ml1 of Fragment C induces significant MIP-1a and MIP-1b
production as early as 3 days post-stimulation (Fig. 1A).
RANTES was present at concentrations higher than MIP-1a
and MIP-1b in unstimulated cells, and antigen stimulation did
not increase its release as much as that of MIP-1a and MIP-1b
(Fig. 1A). The magnitude of the response was comparable
to that to C. albicans antigen (Fig. 1). All the subjects
who displayed proliferative responses (9 out of 12) also
produced chemokines MIP-1a and MIP-1b in response to
Fragment C. A positive response was defined as a quantity of
chemokine at least four standard deviations above the median
of the levels measured in unstimulated cultures. Our data show
that while chemokine release can be measured reliably at day
3 post-stimulation, proliferative responses are not measurable
at that time point (Fig. 1A and B), except when the polyclonal
stimulator PHA is used (data not shown). Therefore, recall
antigen stimulation can be easily measured by chemokine
release at time points where little or no cell proliferation is
detectable. Importantly, not all chemokines are induced by
antigen stimulation. We tested the same supernatants by ELISA
for the presence of two other CC chemokines, macrophagederived chemokine (MDC) and I309. Our results indicate that
MDC production is significantly delayed as compared with the
production of CCR5 ligands, as MDC is barely detectable on
day 3, while its concentration rises on day 6 and, more robustly
on day 9 (Fig. 2). In contrast, we could not observe significant
increase of I309 production by any stimulus, including PHA
(Fig. 2). Therefore, CCR5 ligands release is detectable at time
points when little or no proliferation is detectable.
Kinetics of release of CCR5 ligands
We evaluated the kinetics of release of CCR5 ligands, and
found that MIP-1a and MIP-1b are released very early upon
antigen activation. A first set of experiments (Fig. 3A) revealed
that these chemokines were released as early as 1 day after
activation. When we evaluated the production of CCR5 ligand
within 1 day of stimulation, we found that MIP-1a and MIP-1b
production can be detected as early as 2 h after TTC stimulation, becomes very prominent at 4–6 h and peaks by 16–20 h
(Fig. 3B and C). This suggests that chemokine release is a very
early event in the consequence of stimulation by recall
antigens.
Production of other cytokines
Since our observation on CCR5 ligands release suggests that
these chemokines may be a very sensitive marker of antigen
Fig. 1. Production of chemokines versus [3H]TdR uptake following antigen stimulation of PBMC at day 3 (A) and day 6 (B) with TTC or Candida
albicans. PBMC (1 3 106 for each activation group) were obtained from healthy donors and stimulated with antigen indicated as described in
Methods. Chemokines were detected by a commercial ELISA. [3H]TdR uptake assays were carried on for 6 h. N ¼ 12. Bars indicate standard error
of the mean.
1626 CCR5 ligands release in immune memory responses
Fig. 2. Production of MDC and I309 following stimulation with TTC or Candida albicans at days 3, 6 and 9 after stimulation. PBMC (1 3 106 for
each activation group) were obtained from healthy donors and stimulated with antigen indicated as described in Methods. Chemokines were
detected by commercial ELISA. N ¼ 4. Bars indicate standard error of the mean.
activation, we investigated the release of cytokines that have
classically been linked to immune response, namely, IL-2, IL-4
and IFN-c. We could not detect significant release of IL-4 in
response to antigen (TTC and C. albicans) activation (data not
shown) by ELISA, at the same dilution factor (1/5) as in the
case of CCR5 ligands assays. We were able to detect the
release of IFN-c in five (31.2%) out of the 16 donors described
above and IL-2 in two (12.5%) of the 16 donors (data not
shown) in response to TTC activation. This is in contrast with
detectable levels of CCR5 ligands observed in 13 (81.2%) of
the same 16 donors. In comparison, when we stimulated cells
with C. albicans we detected the release of IFN-c in seven of
the 16 donors, while MIP-1a and MIP-1b release was detected
in 14 (87.5%) of the 16 donors (IL-2 release was not detectable
in any donor with this stimulus).
Chemokine release as a function of time from vaccination
We wanted to establish whether the responses that we
observed diminish in magnitude with time from vaccination.
We analyzed chemokine production and proliferation (3, 6
and 9 days after stimulation) in four subjects who had not received a tetanus booster in the past 10 years. All these
subjects responded to activation by producing chemokines
(Fig. 4). By contrast, antigen-specific proliferation was not
detectable at any of the time points tested for any of these
volunteers (Fig. 4, data not shown). Therefore, it appears that
chemokine secretion is a more reliable indicator of long-term
memory to TTC than T cell proliferation.
Antigen-induced chemokine release can be measured in
cryopreserved samples
The results presented above were obtained using freshly
harvested PBMC. Since sometimes freshly obtained samples
are not available for studies, it was important to determine
whether our assay is effective when cryopreserved specimens
are used. This was determined using PBMC from healthy
donors (N = 12). One aliquot of cells was evaluated for
chemokine secretion as described above, while another aliquot
was cryopreserved in liquid nitrogen for 4 months. At this time
the cells were thawed and stimulated with 3, 10 or 20 lg of TTC,
and with C. albicans as control. Cultures were established with
media only to provide a negative control. Aliquots of supernatants were harvested at days 3, 6 and 9, and tested for
MIP-1a and MIP-1b release by ELISA. On the same days,
proliferation was measured by [3H]TdR uptake. Proliferative
responses to antigen stimulation could be measured on day 6 in
fresh but not cryopreserved cells (data not shown). MIP-1a, but
not MIP–1b (Fig. 5), release was significantly elevated upon
TTC stimulation over control values in both fresh and cryopreserved samples. Release of both MIP-1a and MIP-1b was
significantly increased upon C. albicans stimulation. RANTES
production was also increased. We observed a higher background production and variability of antigen-induced chemokine release in cryopreserved samples compared with freshly
obtained cells; nonetheless, we show that MIP-1a can be
reliably measured in cryopreserved samples, thus enabling its
study in samples obtained from repositories.
Release of CCR5 ligands in the presence of transcription,
translation and Golgi apparatus inhibitors
In order to gain some insight into the mechanism of the prompt
release of CCR5 ligands, we performed activation experiments
with TTC, alone or in the presence of inhibitors of transcription
(actinomycin D, 5 lg ml1, which inhibits both DNA and DNA
polymerization; with short-term treatment it can be assumed
that its action is directed mostly against RNA polymerases),
translation inhibitors (cycloheximide, 2.5 lg ml1) or posttranslational processing inhibitors (BFA, 5 lg ml1, a blocker of
transport from endoplasmic reticulum to Golgi apparatus).
These studies showed that the early release (2–6 h) of CCR5
ligands MIP-1a (Fig. 6A) and MIP-1b (Fig. 6B) is dramatically
inhibited by BFA, while treatment with transcriptional and
translational inhibitors inhibited the release of these chemokines to a much lesser extent, especially at the earlier time
CCR5 ligands release in immune memory responses 1627
Fig. 4. Production of chemokines versus [3H]TdR uptake following
antigen stimulation at day 3 from PBMC of subjects vaccinated > 10
years prior to testing. PBMC (1 3 106 for each activation group) were
obtained from healthy donors and stimulated with TTC or with Candida
albicans as described in Methods. N ¼ 4. Bars indicate standard error
of the mean.
Fig. 3. Time course production of chemokine production in response
to antigen activation. PBMC (1 3 106 for each activation group) were
obtained from healthy donors and stimulated with antigen indicated
as described in Methods. (A) Production of RANTES, MIP-1a and
MIP-1b between days 1 and 3 after stimulation. (B) Production of MIP1a, and (C) production of MIP-1b within the first 24 h of stimulation. N ¼ 4 (different donors were used for the experiments). Bars
indicate standard error of the mean.
points. This suggests that the earlier phases of release of CCR5
ligands are due to a pre-existing intracellular accumulation of
the proteins, while transcription and translation contribute at
a later time to the production of these chemokines.
Flow cytometric analysis of cells releasing
the CCR5 ligand MIP-1b
We investigated the cell types involved in the antigen
activation protocol that we used in this study. We have set
up a protocol for intracellular staining with MIP-1b antibodies
(see Methods) (23). After stimulation for 6 h with TTC and SEB
and blockade of Golgi processing with BFA for 5 h, we stained
cells with fluorescein-conjugated antibodies specific for MIP1b, in conjunction with other markers including CD3 (labeled
with PerCP), CD4 (labeled with APC), IFN-c (labeled with PE)
or CD69 (labeled with PE). Gates were imposed to define
lymphocyte population based on cell size and expression of
CD3 and CD4. Our analyses indicate that following TTC
activation, both CD4+ and CD4 T cells (putatively ascribed as
Fig. 5. Release of CCR5 ligands MIP-1a and MIP-1b from cryopreserved cells. PBMC (1 3 106 for each activation group) were obtained from 12 healthy donors. An aliquot of the cells used in Fig. 1
were cryopreserved in liquid nitrogen for 4 months, and then thawed
and stimulated. Cryopreserved cells were stimulated with 3, 10 or
20 lg of TTC, or cultured with media only, as a control. Aliquots of
supernatants were harvested at days 3, 6 and 9, and tested for MIP-1a
and MIP-1b release by ELISA. Bars indicate standard error of the
mean.
CD8+ T cells) displayed augmented intracellular staining
for MIP-1b, as compared with unstimulated control cells
(Fig. 7—cells in the right quadrant were considered positive).
On the contrary, as described previously, in SEB-treated cells,
the CD4 sub-population showed the most dramatic increase
in MIP-1b intracellular staining (23). We did not observe a
clear-cut association between IFN-c and MIP-1b secretion, or
between MIP-1b and the activation marker CD69 (Fig. 7).
CCR5 ligands in supernatants from antigen-activated cells
inhibit infection by R5 tropic HIV-1 in vitro
In order to test whether CCR5 ligands released upon antigen
activation of PBMC with recall antigens could inhibit HIV
1628 CCR5 ligands release in immune memory responses
Fig. 6. Antigen activation induces early release of MIP-1a (A) and MIP-1b (B), likely because of pre-existing intracellular storage. A total of 1 3 106
PBMC were activated with 10 lg ml1 of TTC as described in the text, and incubated with cycloheximide (2.5 lg ml1), actinomycin D (5 lg ml1)
or BFA (5 lg ml1). Chemokine production in tissue culture supernatants was measured by ELISA at 2 and 6 h after stimulation.
Fig. 7. Flow cytometric analysis of activated cells. Cells in whole blood were activated with 10 lg ml1 of TTC and SEB as described in Methods,
treated with BFA and stained with fluorescent labeled antibodies against MIP-1b, IFN-c, CD4 and CD3. Contour plots of a representative
experiment are shown in (A), while average percentages of positive cells of three experiments are shown in (B). Bars indicate standard error of the
mean.
replication, we set up an in vitro infectivity assay using PHAactivated PBMC (1 3 105 cells per well), which were infected
for 2 h with 100 TCID50 of HIVBaL, an R5 isolate, and then
washed three times and complete media added (25 and 50%
of supernatants from cells activated with TTC). As a control, we
also used supernatants from cells that had not been activated,
and we also infected PBMC without treatment with supernatants from antigen activation. We tested supernatants
obtained from cells from five different donors and harvested
at 8 or 24 h after activation. In some cases, we tested the
supernatants in the presence or absence of a cocktail of
neutralizing polyclonal IgG (Nab) against CCR5 ligands
(100 lg ml1 each of anti-RANTES, anti-MIP-1a and antiMIP-1b, R&D Systems). We observed that supernatants from
antigen-activated cells, at both 25% (Fig. 8) and 50% (data
not shown) concentrations, reproducibly inhibited HIVBaL
replication, as measured by the release of p24 in the tissue
culture supernatants between days 4 and 10 after infection
(Fig. 8). Interestingly, supernatants from unstimulated cells
could also inhibit HIV replication, possibly due to background
release of CCR5 ligands, especially RANTES, but the inhibition
was lower than that observed using supernatants from
activated cells. However, when supernatants were pre-treated
for 1 h with Nab to CCR5 ligands prior to being added to the
infectivity assays, we observed a complete reversal of the
inhibition of HIV replication (Fig. 8), which suggests that all
the inhibitory activity that we observed in this system was due
to CCR5 ligands release.
Discussion
Chemokines, by virtue of their ability to attract discrete
leukocyte sub-populations, are key mediators of immune
response. Their concerted release is the basis of migration of
CCR5 ligands release in immune memory responses 1629
Fig. 8. Supernatants from antigen-activated cells inhibit R5 HIV
replication. Supernatants from PBMC after 8 h of activation were
added to 1 3 105 cells that were infected for 2 h with 100 TCID50 of
HIVBaL. Some of the supernatants were added with 100 lg each of
antibodies against RANTES, MIP-1a and MIP-1b, or with an irrelevant
IgG. Cells were then washed three times with PBS, and incubated in
the presence of supernatants for up to 10 days. HIV replication was
monitored by p24 ELISA. Shown are results from a representative
experiment out of five independent experiments performed using
supernatants obtained from different donors.
cells from the periphery to a site of inflammation or infection
(24). In this study, we investigated the relevance of CCR5
ligands in the context of the immune response triggered by
a protein antigen, i.e. TTC. We selected these chemokines
since they bind to the HIV co-receptor most involved in
primary HIV infection, as demonstrated by the protection from
HIV infection conferred by the D32CCR5 mutant allele in the
homozygous state (25–30). We (10, 31) and others (11, 12,
15–19, 32–35) had previously shown that high levels of chemokine release from antigen-activated cells are associated with
protection from HIV-1 infection in high-risk subjects, and with
better disease outcome in HIV-infected individuals. Furthermore, studies on non-human primates that were immunized
against SIV showed that protection was related to high levels
of CCR5 release from cells (36–40). These findings were
consistent with our hypothesis (41) that release of high levels
of chemokines may constitute a correlate of protection from
viruses that use CCR5 as a receptor to enter target cells (4–8),
i.e. HIV and simian immunodeficiency virus (SIV). Several
studies have shown that CCR5 ligands are released in the
course of immune response to a cognate antigen (10–14, 19,
34, 36, 37). This is of great importance from the viewpoint of
developing effective AIDS vaccines, as release of HIV
inhibitory chemokines would add an additional line of defense
to the classical two arms of the immune response, i.e.
neutralizing antibodies and CTL. Therefore, to study the
release of CCR5 ligands from blood cells, we chose to study
the immune response to an antigen that is the functional
equivalent of a subunit vaccine, i.e. TTC, reasoning that an
effective HIV vaccine should be at least as capable of
triggering an immune response as this experimental antigen.
Our results showed that CCR5 ligands release occurs
promptly after antigen stimulation, as significant levels of
these chemokines, except RANTES, which was measured in
supernatants even without simulation, were reached as early
as 2 h after activation, and started peaking at around 8 h. The
release of MIP-1a and MIP-1b was detectable well before
proliferative responses, as measured by uptake of [3H]TdR,
which requires 6 days of incubation. Such fast kinetics of
release occur in the same time frame as the events required for
HIV entry (21–22), therefore enabling CCR5 ligands, in
principle, to interfere with HIV infection. The fast kinetics
of response to antigen suggests that it could be advantageous
to measure CCR5 ligands, as compared with measuring
lymphocyte proliferation, when responses to vaccines are
measured in developing countries, by reducing the amount of
equipment required for testing and the time needed for
incubation and by eliminating the need for radioactivity.
Furthermore, release of CCR5 ligands was found to be a more
sensitive marker of immune response to recall antigen than
cell proliferation measured by thymidine incorporation, since
cells obtained from individuals that had been immunized
against tetanus > 10 years prior to testing did not display
any detectable proliferative responses to TTC stimulation,
while CCR5 ligands release was consistently found to be
elevated. Interestingly, we observed that CCR5 ligands
were released in a specific manner in response to antigen
stimulation, since other chemokines, which also inhibit HIV (42,
43), were not elevated, as in the case of I309, or were released
much later in time, like MDC (Fig. 2). Flow cytometric analysis
of cells secreting MIP-1b revealed that both CD4+ and CD8+
T lymphocytes are involved in the release of this CCR5 ligand
in response to TTC. We observed that the percentage of
cells that release MIP-1b is comparable to the percentage of
cells producing IFN-c, under our experimental conditions.
Similar findings were reported by us and others (23, 44)
using different stimuli and/or selected cell populations. Thus,
it appears that even in flow cytometric analyses, MIP-1b is
a marker at least as powerful as IFN-c in detecting cell
activation by antigen stimulation.
When we analyzed the mechanism of release of CCR5
ligands, we observed that the early phase of release can be
inhibited by BFA, which interferes with the processing of
proteins in the Golgi apparatus, while inhibitors of transcription
and translation had limited efficacy in blocking early CCR5
release. Thus, it appears that the initial release of CCR5
ligands occurs independently of transcription and translation,
and is therefore likely to be due to the release of these
chemokines from a pre-existing accumulation, which could
explain the fast kinetics of this phenomenon. Recent data on
memory and effector CD8+ T cells support this concept,
having demonstrated that RANTES release occurs in a quick
burst within an hour following antigen activation (45, 46).
RANTES production in these conditions appears to be due to
a pre-existing accumulation of the protein in the cytoplasm,
unlike the release of IFN-c, which occurs later and is probably
due to a transcriptional mechanism (46). The kinetics of
RANTES release and its resistance to cycloheximide treatment
(47) are very similar to our observations, thus supporting the
finding that CCR5 ligands are released as a very early
response to antigen activation.
We determined that supernatants from antigen-activated
cells could inhibit infection by an R5 isolate of HIV-1. This
inhibition was reversed by the addition of anti-CCR5 ligands
antibodies, indicating that the bulk of the specific anti-R5 HIV
1630 CCR5 ligands release in immune memory responses
activity in these supernatants is due to these chemokines in
accordance with our previous studies (10, 31). Since CCR5
ligands can attract CD4+ T cells, it is conceivable that their
release upon antigen activation may contribute to, rather than
inhibiting, infection of cells (47–49). However, the vast majority
of clinical studies suggests that the balance between protection from infection and increased availability of uninfected
cells, if existing, is tilted toward protection (10–13, 15–19, 31–
35). It should be noted that most of the literature suggesting
that chemokines might be a promoting factor in HIV infection
were performed using plasma and serum samples (50–56),
which yield inherently inaccurate chemokine measurements,
vis-à-vis the non-circulating nature of these molecules and
their variable levels in these fluids due to the chemokines
ability to aggregate and their release from platelets (57–61).
Thus, in the case of immunization against HIV, it is conceivable
that the release of CCR5 ligands could play a role in protection.
While it is true that vaccination against pathogens other than
HIV has been associated with temporary increases in viremia
in HIV-infected subjects (62), a different scenario might occur
with HIV immunization. In our hypothetical model, we envision
that HIV, which preferentially infects HIV-specific cells (20),
would stimulate a memory response, in individuals immunized
against it, which in its early phase would include CCR5 ligands
release. This release would prevent HIV entry into target cells,
since most primary transmission occurs through R5 strains.
Therefore, the overall effect of immune stimulation and CCR5
ligands release should be evaluated in the context of HIV
immunization. In support of this hypothesis, several published
studies have linked protection from SIV infection to levels of
CCR5 ligands in an immunization animal model (36–40). Thus,
further studies on the release of CCR5 ligands in the context of
HIV vaccination are warranted.
In summary, our data show that CCR5 ligands MIP-1a and
MIP-1b constitute one of the most sensitive markers of memory response, and are released in quantities and kinetics
compatible with inhibition of HIV infection in vitro. Consequently, their study is inescapable in the context of vaccination, toward the goal of identifying immunogens that can
protect against HIV infection.
Abbreviations
AIDS
APC
BFA
c.p.m.
[3H]TdR
HIV
MDC
MIP
PerCP
RT
SDF
SEB
SIV
TTC
acquired immunodeficiency syndrome
allophycocyanin
brefeldin A
counts per minute
tritiated thymidine
human immunodeficiency virus
macrophage-derived chemokine
macrophage inflammatory protein
peridinin-chlorophyll protein
room temperature
stromal cell derived factor
Staphylococcus aureus enterotoxin B
simian immunodeficiency virus
Fragment C of tetanus toxin
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