The integrin alpha4beta7 forms a complex with cell-surface CD4 and defines a T-cell subset that is highly susceptible to infection by HIV-1.
ABSTRACT Both activated and resting CD4(+) T cells in mucosal tissues play important roles in the earliest phases of infection after sexual transmission of HIV-1, a process that is inefficient. HIV-1 gp120 binds to integrin alpha(4)beta(7) (alpha(4)beta(7)), the gut mucosal homing receptor. We find that alpha(4)beta(7)(high) CD4(+) T cells are more susceptible to productive infection than are alpha(4)beta(7)(low-neg) CD4(+) T cells in part because this cellular subset is enriched with metabolically active CD4(+) T cells. alpha(4)beta(7)(high) CD4(+) T cells are CCR5(high) and CXCR4(low); on these cells, alpha(4)beta(7) appears in a complex with CD4. The specific affinity of gp120 for alpha(4)beta(7) provides a mechanism for HIV-1 to target activated cells that are critical for efficient virus propagation and dissemination following sexual transmission.
- SourceAvailable from: Jean-Charles Grivel[Show abstract] [Hide abstract]
ABSTRACT: To enter target cells HIV-1 uses CD4 and a coreceptor. In vivo the coreceptor function is provided either by CCR5 (for R5) or CXCR4 (for X4 HIV-1). Although both R5 and X4 HIV-1 variants are present in body fluids (semen, blood, cervicovaginal and rectal secretions), R5 HIV-1 appears to transmit infection and dominates early stages of HIV disease. Moreover, recent sequence analysis of virus in acute infection shows that, in the majority of cases of transmission, infection is initiated by a single virus. Therefore, the existence of a "gatekeeper" that selects R5 over X4 HIV-1 and that operates among R5 HIV-1 variants has been suggested. In the present review we consider various routes of HIV-transmission and discuss potential gatekeeping mechanisms associated with each of these routes. Although many mechanisms have been identified none of them explains the almost perfect selection of R5 over X4 in HIV-1 transmission. We suggest that instead of one strong gatekeeper there are multiple functional gatekeepers and that their superimposition is sufficient to protect against X4 HIV-1 infection and potentially select among R5 HIV-1 variants. In conclusion, we propose that the principle of multiple barriers is more general and not restricted to protection against X4 HIV-1 but rather can be applied to other phenomena when one factor has a selective advantage over the other(s). In the case of gatekeepers for HIV-1 transmission, the task is to identify them and to decipher their molecular mechanisms. Knowledge of the gatekeepers' localization and function may enable us to enhance existing barriers against R5 transmission and to erect the new ones against all HIV-1 variants.Journal of Translational Medicine 01/2011; 9 Suppl 1:S6. · 3.46 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: It is well established that HIV-1 infection typically involves an interaction between the viral envelope protein gp120/41 and the CD4 molecule followed by a second interaction with a chemokine receptor, usually CCR5 or CXCR4. In the early stages of an HIV-1 infection CCR5 using viruses (R5 viruses) predominate. In some viral subtypes there is a propensity to switch to CXCR4 usage (X4 viruses). The receptor switch occurs in ~ 40% of the infected individuals and is associated with faster disease progression. This holds for subtypes B and D, but occurs less frequently in subtypes A and C. There are several hypotheses to explain the preferential transmission of R5 viruses and the mechanisms that lead to switching of co-receptor usage; however, there is no definitive explanation for either. One important consideration regarding transmission is that signaling by R5 gp120 may facilitate transmission of R5 viruses by inducing a permissive environment for HIV replication. In the case of sexual transmission, infection by HIV requires the virus to breach the mucosal barrier to gain access to the immune cell targets that it infects; however, the immediate events that follow HIV exposure at genital mucosal sites are not well understood. Upon transmission, the HIV quasispecies that is replicating in an infected donor contracts through a "genetic bottleneck", and often infection results from a single infectious event. Many details surrounding this initial infection remain unresolved. In mucosal tissues, CD4(+) T cells express high levels of CCR5, and a subset of these CD4(+)/CCR5(high) cells express the integrin α₄β₇, the gut homing receptor. CD4(+)/CCR5(high)/ α4β7(high) T cells are highly susceptible to infection by HIV-1 and are ideal targets for an efficient productive infection at the point of transmission. In this context we have demonstrated that the HIV-1 envelope protein gp120 binds to α₄β₇ on CD4(+) T cells. On CD4(+)/CCR5(high)/ α4β7(high) T cells, α₄β₇ is closely associated with CD4 and CCR5. Furthermore, α₄β₇ is ~3 times the size of CD4 on the cell surface, that makes it a prominent receptor for an efficient virus capture. gp120-α₄β₇ interactions mediate the activation of the adhesion-associated integrin LFA-1. LFA-1 facilitates the formation of virological synapses and cell-to-cell spread of HIV-1. gp120 binding to α₄β₇ is mediated by a tripeptide located in the V1/V2 domain of gp120. Of note, the V1/V2 domain of gp120 has been linked to variations in transmission fitness among viral isolates raising the intriguing possibility that gp120-α₄β₇ interactions may be linked to transmission fitness. Although many details remain unresolved, we hypothesize that gp120-α₄β₇ interactions play an important role in the very early events following sexual transmission of HIV and may have important implication in the design of vaccine strategies for the prevention of acquisition of HIV infection.Journal of Translational Medicine 01/2011; 9 Suppl 1:S2. · 3.46 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: T lymphocyte dysfunction contributes to human immunodeficiency virus type 1 (HIV-1) disease progression by impairing antivirus cellular immunity. However, the mechanisms of HIV-1-infection-mediated T cell dysfunction are not completely understood. Here, we provide evidence that expansion of monocytic myeloid-derived suppressor cells (M-MDSCs) suppressed T-cell function in HIV-1-infected individuals. We observed a dramatic elevation of M-MDSCs (HLADR(-/low)CD11b(+)CD33(+/high) CD14(+)CD15(-) cells) in the peripheral blood of HIV-1-seropositive subjects (n = 61) compared with healthy controls (n = 51), despite efficacious antiretroviral therapy for nearly 2 years. The elevated M-MDSC frequency in HIV-1(+) subjects correlated with prognostic HIV-1 disease markers, including HIV-1 viral load (r = 0.5957, p < 0.0001), CD4(+) T cell loss (r = -0.5312, p < 0.0001), and activated T cells (r = 0.4421, p = 0.0004). Functional studies showed that M-MDSCs from HIV-1(+) subjects suppressed T cell responses in both HIV-1-specific and antigen-nonspecific manner, this effect was dependent on the induction of arginase-1 and required direct cell-cell contact. Further investigations revealed that direct HIV-1 infection or culture with HIV-1-derived Tat protein significantly enhanced human MDSC generation in vitro, and MDSCs from healthy donors could be directly infected by HIV-1 to facilitate HIV-1 replication and transmission, indicating a positive feedback loop between HIV-1 infection and MDSC expansion existed. In summary, our studies revealed a novel mechansim of T cell dysfunction in HIV-1-infected individuals, and suggested that targeting MDSCs may be a promising strategy for HIV-1 immunotherapy.Journal of Virology 11/2012; · 5.08 Impact Factor
The integrin ?4?7forms a complex with cell-surface
CD4 and defines a T-cell subset that is highly
susceptible to infection by HIV-1
Claudia Cicalaa,1,2, Elena Martinellia,1, Jonathan P. McNallya,1, Diana J. Goodea, Ravindra Gopaula, Joseph Hiatta,
Katija Jelicica, Shyamasundaran Kottilila, Katilyn Macleoda, Angeline O’Sheaa, Nikita Patela, Donald Van Ryka,
Danlan Weia, Massimiliano Pascuccioa, Ling Yib, Lyle McKinnonc, Preson Izullad, Joshua Kimanid, Rupert Kaulc,
Anthony S. Faucia,2, and James Arthosa
aLaboratory of Immunoregulation andbLaboratory of Molecular Immunology, National Institute of Allergy and Infectious Diseases, National Institutes of
Health, Bethesda, MD 20892;cDepartment of Medicine, University of Toronto, Toronto, Canada M5S 1A8; anddDepartment of Medical Microbiology,
University of Nairobi, P.O. Box 30197-00100, Kenya
Contributed by Anthony S. Fauci, October 15, 2009 (sent for review September 18, 2009)
Both activated and resting CD4?T cells in mucosal tissues play
important roles in the earliest phases of infection after sexual trans-
mission of HIV-1, a process that is inefficient. HIV-1 gp120 binds to
integrin ?4?7(?4?7), the gut mucosal homing receptor. We find that
are ?4?7low-negCD4?T cells in part because this cellular subset is
enriched with metabolically active CD4?T cells. ?4?7highCD4?T cells
are CCR5highand CXCR4low; on these cells, ?4?7appears in a complex
with CD4. The specific affinity of gp120 for ?4?7provides a mecha-
nism for HIV-1 to target activated cells that are critical for efficient
virus propagation and dissemination following sexual transmission.
integrin receptor ? transmission ? gut-associated lymphoid tissues (GALT)
?4?7facilitates the migration of lymphocytes from gut-inductive
sites where immune responses are first induced (Peyer’s patches
and mesenteric lymph nodes) to the lamina propria (1, 2). These
three gut-associated lymphoid tissues (GALT) play central roles in
the initial phases of infection following sexual transmission. Anti-
gen-specific ?4?7?CD4?T cells are also found in genital mucosa
(3, 4), where CD4?T cells are first infected (5, 6). Within days
following sexual transmission, infected cells migrate from the
genital mucosa to Peyer’s patches and mesenteric lymph nodes
where high-level HIV replication occurs (5). HIV also enters the
lamina propria where it mediates a massive depletion of CD4?T
cells (7). Both resting and activated CD4?T cells play key roles
transmission and the early stages of infection remain unknown.
Understanding these events may prove vital to the development of
an effective vaccine. It is clear, however, that ?4?7is functionally
linked to each of the sites involved in the earliest phases of acute
infection. It is in this context that we recently described a specific
and ?4?7on CD4?T cells (9). As a result, virions are captured by
?4?7 on the surface of CD4?T cells. Unlike the HIV entry
receptors (CD4 and CCR5), ?4?7is not required for viral replica-
mesenteric lymph nodes, lamina propria and the earliest phases of
acute infection, suggests that gp120- ?4?7 interaction plays an
important role at an early point in the HIV infection cycle in vivo.
In support of this proposition, we determined that ?4?7reactivity
we tested. Moreover, ?4?7 binding is mediated by a conserved
tripeptide in the gp120 V2 loop that mimics tripeptides presented
in MadCAM, VCAM, and fibronectin, the three natural ligands of
?4?7. Both the conserved nature of this interaction and the evident
molecular mimicry implies that engaging ?4?7provides a selective
advantage to HIV-1.
Sexual transmission and the establishment of HIV infection in a
new host is inefficient. This is evidenced by studies demonstrating
that, in discordant couples, multiple exposures typically are re-
quasi-species replicating in an infected donor contracts through a
‘‘genetic bottleneck’’ upon transmission, and infection often ap-
pears to result from a single infectious event (11), suggesting that
abortive infections following sexual transmission may occur fre-
In this report, we demonstrate that ?4?7demarcates a distinct
subset of CCR5high/CD4?T cells that are prone to productive
acquired an affinity for ?4?7as a means of targeting these highly
susceptible cells, thereby increasing the likelihood of establishing
productive infection following sexual transmission.
?4?7highCD4?T Cells Are Prone to Highly Productive HIV Infection.
?4?7is activated and expressed at high levels on CD4?T cells in
immune-competent tissues of the gut that are undergoing antigen
specific activation (12). In contrast, only a minor subset of freshly
majority of which appears on the cell membrane in an inactive
conformation. However, by culturing peripheral CD4?T cells in
a gut phenotype that includes increased ?4?7expression (13). Such
cultures include CD4?T-cell subsets that can be described as
?4?7high, ?4?7low, and ?4?7negative. Under the conditions we employ,
expression of integrin ?7(Fig. S1). To better understand the role of
included ?4?7high, ?4?7low, and ?4?7negativecells with HIV-1 SF162,
a CCR5-tropic isolate, and virus production was monitored by
intracellular staining of HIV-1 Gag p24. Three days post-infection
an ?4?7highphenotype (Fig. 1A). The increased production of p24
in ?4?7highcells occurred over a range of virus inputs (Fig. 1B and
Fig. S2). Over time the ?4?7highcells disappeared and viral pro-
duction shifted to ?4?7lowand ?4?7negativesubsets (Fig. 1A). The
C.C., E.M., J.P.M., A.S.F., and J.A. wrote the paper.
The authors declare no conflict of interest.
Freely available online through the PNAS open access option.
1C.C., E.M., and J.P.M. contributed equally to this work.
whom correspondencemaybe addressed. E-mail:email@example.com or
This article contains supporting information online at www.pnas.org/cgi/content/full/
December 8, 2009 ?
vol. 106 ?
no. 49 ?
selective depletion of ?4?7highcells in infected cultures was striking
expression of CD45RO on ?4?7highcells and found that it also
disappeared (Fig. S3). It is unlikely that HIV-1 mediates the
downregulation of both receptors; therefore, we conclude that the
loss of ?4?7highCD4?T cells was the result of virus-mediated cell
death. To confirm the bias toward HIV-1 replication in ?4?7high
cells we separated the ?4?7highcellular subset from the ?4?7low-neg
subsets (Fig. 1D, inset) and then inoculated cultures with a low
amount of virus (100? less than in Fig. 1A). Viral replication was
monitored by p24 antigen ELISA. On the peak day of replication
in ?4?7highcells from three donors we observed an average of
15-fold greater level of replication in the ?4?7highsubset than in the
?4?7low-negsubset (P ? 0.027, Wilcoxon rank-sum test) (Fig. 1D).
?4?7highCD4?T Cells Are CCR5highand Metabolically Activated. To
better understand the preferential replication of HIV-1 in ?4?7high
cells, we carried out additional phenotypic analyses. ?4?7highcells
are CD45RO?, and unlike the ?4?7lowand ?4?7negcells expressed
high levels of CCR5 (Fig. 2 A and B). ?4?7highcells were strongly
reactive for Ki-67, indicative of an activated metabolic state (Fig.
2C). These cells were also distinct in expressing low levels of
CXCR4 and could be further defined as CD45RA?/CD62L?/
CCR7?, consistent with a memory phenotype (Fig. S4). We
conclude that the rapid infection and high-level production of
HIV-1 in ?4?7highCD4?T cells can be explained by the fact that
these cells are metabolically activated, although other factors may
also contribute. Thus, high levels of ?4?7demarcate a subset of
CCR5high/CD4?T cells that are an ideal substrate for highly
productive HIV-1 infection.
Activated CD4?T Cells Occur at a High Frequency in the ?4?7high
Subset of Gut Mucosal CD4?T Cells. The fact that HIV-1 has the
capacity to bind to a receptor that defines a subset of activated
targets provides potentially important clues about how HIV-1 uses
?4?7in vivo. To that end, we determined whether ?4?7highCD4?T
cells taken directly from gut tissues exhibited a similar phenotype.
Practical limitations confined our analysis to rectum and colon
from three healthy donors, the frequency of ?4?7highCD4?T cells
also CCR5highand Ki-67?(95% CI 18–28%) (Fig. 3B). In contrast,
were CCR5highand Ki-67?. Therefore, the percentage of Ki-67?
cells is higher in the ?4?7highsubset than in the ?4?7low-negsubset.
Thus, Ki-67?cells were enriched in the ?4?7highCD4?cell subset
both in vitro (Fig. 2) and ex vivo (Fig. 3).
?4?7?CD4?T Cells Reside in Female Genital Mucosa. The initial
events that follow HIV deposition in genital mucosal sites are
poorly understood, as are the events that promote HIV-1 invasion
of GALT. In considering whether ?4?7plays a role in infection of
Day 3Day 6 Day 8
Day 3 Post Infection
%p24+ cells in each population
13 28 18
p24 ELISA (ng/mL)
Day 3 Day 6Day 8
% of Max
of productive infection. In vitro infection of CD4?T
cells after 6 days of culture in the presence of retinoic
acid. (A) Flow cytometric analysis of cell surface inte-
grin ?7and intracellular Gag p24 expression on and in
CD4?T cells 3, 6, and 8 days after inoculation with the
R5 HIV-1 SF162. (B) Percent intracellular Gag p24 ex-
pression in ?4?7highand ?4?7low-negCD4?T cells 3 days
post-inoculation using three different amounts of
SF162. Values represent the % p24?cells within each
subset. (C) Comparison of integrin ?7expression on
natants harvested from ?4?7highand ?4?7low-negCD4?
T-cell subsets 3, 6, and 8 days post-inoculation with
SF162. Levels of integrin ?7expression on both subsets
on the day of inoculation are included (Inset). These
iments using different donor CD4?T cells.
?4?7highCD4?T cells are a preferential target
CCR5 and are metabolically active. Flow cytometric
analysis of peripheral CD4?T cells cultured in retinoic
acid for 6 days followed by staining with fluorescein-
ated mAbs specific for (A) ?7and CD45RO. (B) ?7and
CCR5. (C) ?7and Ki-67. This is a representative analysis
of receptor expression on more than three indepen-
dent donor CD4?T cells.
?4?7highCD4?T cells express high levels of
www.pnas.org?cgi?doi?10.1073?pnas.0911796106Cicala et al.
T cells in genital mucosa and/or seeding of GALT, we noted a
recent report that described a unique population of ?4?7?memory
T lymphocytes resident in the female reproductive tract (14). This
finding prompted us to determine whether we could identify ?4?7?
CD4?T-cells in female genital mucosa tissue samples. To this end,
we analyzed cervical cytobrush samples obtained from eight
women visiting a female sex worker clinic in Nairobi, Kenya. The
mAb Act-1 was for used to measure the surface expression of ?4?7.
?4?7?CD4?T-cells were detected in all eight samples (mean
17.5%) (Fig. 3C). The presence of these cells could provide a link
between productive infection of CD4?T cells in female genital
mucosa and the subsequent spread of HIV-1 to GALT.
CD4?T cells is unknown. It encounters multiple barriers to
successful transmission (15) as it migrates through highly compart-
mentalized mucosal tissues. Moreover, it has been suggested that
dendritic cells facilitate this migration (15). Neither the biopsies
that we analyzed nor our culture systems reflect this complexity;
however, the capacity of ?4?7to capture HIV-1 virions (9) suggests
that it can facilitate infection of activated cells in vitro. To this end,
we inoculated cultures of ?4?7highCD4?T cells with low amounts
of virus in the presence or absence of the ?4 mAb 2B4 that
1A, and unbound virus was rinsed away 3 h post-inoculation. In
through the culture relative to control cultures (Fig. 4) such that on
day 6 post-inoculation, lower levels of extracellular Gag p24 were
observed in 2B4-treated cultures than in control cultures. By day 9
post-infection, the inhibitory activity of 2B4 decreased. These
results indicate that 2B4 slows the spread of virus through a culture
enriched in ?4?7highCD4?T cells.
CD4 and ?4?7Reside in a Complex on the Cell Membrane. Because
previously observed on RA-cultured T cells (9). We stained freshly
isolated cells from gut biopsies with ?4?7, CD4 and CCR5 mAbs
and CCR5. We observed more pronounced CD4 clustering on gut
cells than we typically observe on peripheral T cells (Fig. S6). This
clustering raised the possibility that these receptors exist in close
physical proximity on the membrane surface. The resolution of
standard confocal microscopy (?200 nm) is insufficient to make
Days Post Infection
Days Post Infection
Days Post Infection
Days Post Infection
% 2B4 Inhibition
Donor 1Donor 3
presence of IgG1 or the ?4mAb 2B4 (as indicated). Viral replication was deter-
mined by measuring by p24 Gag levels in culture supernatants on days 3, 6, and
6 and 9 is presented. 2B4 significantly inhibited viral replication on day 6 (P ?
reduction in the level of p24 on day 6 in the infection in presence of 2B4. Error
significantly greater than on day 9 (Wilcoxon signed-rank test).
Delay in HIV-1 replication by an ?4mAb. (A–C) Purified CD4?T cells
% CCR5high/Ki-67+ CD4+ T Cells
Donor 1 Donor 2Donor 3Avg.
% Act-1+ Cells
rectal and colon biopsies were analyzed by flow cytom-
etry. (A) A representative analysis of CCR5 and Ki-67
expression on ?7highand ?7low-negCD4?T cells. (B) Sum-
mary of Ki-67 and CCR5 expression on ?7highand ?7low-
negCD4?T cells isolated from the colon and rectum of
three healthy donors. Values are reported as % within
each population. Average % expression of CCR5 and
(nonparametric Wilcoxon signed-rank test for paired
samples). (C) ?4?7?/CD4?T cells are detected in female
genital mucosa (Mean 17.5, S.D. 13.4).
Cicala et al. PNAS ?
December 8, 2009 ?
vol. 106 ?
no. 49 ?
such a determination. Thus, to determine if ?4?7and CD4 reside
transfer (FRET) (resolution ?10 nm) (16). Cells were stained with
the ?7mAb FIB27 Alexa Fluor 488 (donor) and the CD4 mAb
Leu3A Alexa Fluor 568 (acceptor) (Fig. S7A). Donor and acceptor
fluorescence levels were recorded both before, and following
acceptor-photobleaching. Regions of interest (ROIs) were created
around clusters of ?7and FRET efficiencies were calculated based
upon changes in donor fluorescence subsequent to acceptor pho-
tobleaching. As a positive control we measured FRET efficiencies
generated between two CD4 mAbs, Leu3A Alexa Fluor 568
(acceptor) and OKT4 Alexa Fluor 488 (donor). As a negative
control, we measured FRET efficiencies generated between CD4
and CD43, an abundantly expressed anti-adhesion receptor known
not to cluster with CD4 on activated cells (17). Twelve cells were
analyzed, one of which is presented in Fig. S7A. ROI-3 (Fig. S7A
?7, with an efficiency of ?50%. Upon analyzing up to 19 ROIs on
12 cells, we observed CD4-?7FRET effects of ?34% (Fig. S7B)
(P ? 0.0001 Wilcoxon signed-rank test for paired samples.) By
comparison, we observed CD4-CD4 FRET effects of approxi-
mately 40% and CD4-CD43 FRET effects of ?5%. These results
were confirmed by sensitized-emission FRET (Fig. S8 A and B).
The resolution of FRET is approximately 10 nm; therefore, these
results indicate that CD4 and ?4?7appear on the surface of a cell
in extremely close association.
Considering the proximity of CD4 and ?4?7 we wondered
whether these receptors could be coprecipitated. Because CD4 is
normally recruited into the T-cell receptor complex upon T-cell
activation, its coprecipitation with ?4?7would represent an unex-
pected pairing on the cell membrane. ?4?7highCD4?T cells were
preincubated on ice and then briefly treated with 3,3?-dithiobis-
(sulfosuccinimidylpropionate) (DTSSP), a crosslinking reagent
with a spacer arm of approximately 1.2 nm. Cells were lysed, and
electrophoresed and immunoblotted with ?7 polyclonal antisera
(Fig. 6A). In the presence of DTSSP, ?7coprecipitated with CD4,
indicating that they can reside within ?1.2 nm of each other.
Considering the close proximity of these two receptors, their
relative height, and the estimated diameter of an envelope spike
(18) (Fig. 6B), it is possible that a gp120 trimer could engage ?4?7
and CD4 in a near-simultaneous manner. We do not yet know
whether such dual ligation takes place on the cell surface.
through an activated/extended form of ?4?7 that is present on
?4?7highCD4?T cells. Others have reported that during the acute
phase of SIV infection, peripheral ?4?7highCD4?T cells are
preferentially infected (19), and, in humans, circulating ?4?7?
CD4?T cells are preferentially depleted during the acute phase of
T cells that can be defined by high levels of ?4?7expression are
prone to highly productive HIV-1 infection. Multiple factors are
CD4?T cells. In particular, a large fraction of these cells are
metabolically active as measured by Ki-67, express high levels of
CCR5, and ?4?7resides on the cell membrane in close association
with CD4. We conclude that these factors provided the selective
Sexual transmission of HIV is inefficient (11, 21). Any replication
with the ?4?7mAb Act-1 (red), the CD4 mAb OKT4 (purple), and the CCR5 mAb 2D7 (green) and viewed under a confocal microscope. Unstained and individual
This cell is representative of greater that 60 cells analyzed from four donors.
www.pnas.org?cgi?doi?10.1073?pnas.0911796106Cicala et al.
advantage that HIV-1 can gain during the early stages of infection
can have a significant impact on the efficiency of transmission.
Although resting cells are thought to be the first cells infected,
activated cells are critical to propagation and dissemination follow-
ing sexual transmission (5, 6, 22). In this regard ?4?7mediates the
migration of CD4?T cells between Peyer’s patches, mesenteric
lymph nodes, and lamina propria (12, 23), sites that represent a
‘‘target rich’’ environment for HIV-1. Among all lymphoid tissues
in humans, it is in these tissues that the majority of activated CD4?
T cells reside (24). To the extent that HIV-1 can rapidly gain access
Thus, the specific affinity of gp120 for ?4?7likely reflects the fact
that ?4?7is presented on CD4?T cells that are a desirable target
for productive infection.
The CD4 receptor-independent nature of gp120-?4?7interac-
tions is important in understanding the role that this receptor plays
in infection. As noted above, a subset of ?4?7highCD4?T cells are
distinct in expressing high levels of CCR5 and relatively low levels
of CXCR4 (note that sexual transmission strongly favors R5
viruses). High-level expression of CCR5 on CD4?T cells is, to a
degree, a marker of activation in the gut, and one might consider
then that HIV-1 could use CCR5 as a means of discriminating
between active and resting cells. However, unlike ?4?7,CCR5 is
effectively hidden from HIV until after a virion engages a CD4
receptor. The CD4 receptor exhibits near-uniform expression on
CD4?T cells regardless of whether they are metabolically active or
resting, or whether they are CCR5highor CCR5low. Once a virion
engages CD4 receptors, conformational changes in the envelope
spike effectively commit the virion to infect that cell regardless of
that might facilitate productive infection. In contrast, the interac-
tion of the HIV envelope with ?4?7is CD4-independent. Rather
than being hidden from the virus, as is CCR5, the extended form
of ?4?7is estimated to rise ?20 nM from the cell membrane (25).
By comparison, CD4 rises only approximately 7 nM from the cell
surface (26). Thus, ?4?7represents a structurally prominent gp120-
binding receptor on the surface of a highly susceptible subset of
CD4?T cells that HIV-1 can engage independently of CD4.
We show that ?4?7, CD4, and CCR5 colocalize on the cell
membrane. Moreover ?7is sufficiently close to CD4 that these two
two receptors appear together in a complex on the surface of a
that virions (or envelope spikes on infected cells) are likely to first
encounter the activated/extended form of ?4?7and subsequently
engage CD4. This scenario is consistent with a recent structural
near the apex of the trimeric spike (27). An LDV tripeptide in the
gp120 V2-loop mediates binding to ?4?7(9), placing this critical
contact site at or near the tip of the spike, a position well suited for
an initial engagement with ?4?7. It is noteworthy that such posi-
tioning may provide opportunities for therapeutic or vaccine-
Two issues surrounding the interaction between HIV-1 and
?4?7require further investigation. First, what role does gp120
signal transduction through ?4?7play in infection? Such signals
may facilitate the infection of activated cells but may also
promote infection of suboptimally activated ?4?7CD4?T cells.
In this regard, we have previously reported that among the
subsets of CD4?T cells, the ?4?7highsubset is distinct in
responding to gp120-mediated signals by rapidly activating
LFA-1. This is potentially important because LFA-1 plays a
in the preferential infection of memory CD4?T cells (28). Yet,
the specific sequence of events that follow cell-free or budding
virion engagement of ?4?7 and how those events facilitate
infection remain to be determined. Second, activated/extended
?4?7is found principally in Peyer’s patches, mesenteric lymph
nodes, and lamina propria, all of which play central roles in the
yet know in which of these tissues the interaction between HIV-1
and ?4?7is most relevant. Moreover, ?4?7?CD4?T cells have
also been found in genital mucosa (3, 14), and we detected ?4?7?
CD4?T in cervical cytobrush samples. In this regard, the genital
mucosa does not contain well-organized immune-inductive sites;
instead, it relies upon ?4?7highCD4?T cells trafficking from
more organized immune-inductive sites, including Peyer’s
patches (29). Thus, we cannot exclude the possibility that the
initial and most relevant interaction between HIV-1 and ?4?7
occurs in the genital mucosa in the earliest days following
exposure. Whether this interaction involves free virions, infected
cells, or virions sequestered by dendritic cells (15, 30) remains to
In conclusion, we have demonstrated that ?4?7demarcates a
In rectum and colon biopsies, the highest frequency of metaboli-
cally activated CD4?T cells appears in the ?4?7highpopulation.
Although there are other markers of activation on CD4?T cells,
in close physical proximity to the CD4 receptor and because it
mediates the trafficking of CD4?T cells to the gut tissues that are
an ideal environment to firmly establish infection after sexual
transmission. Noting that sexual transmission occurs inefficiently,
we propose that, by interacting with ?4?7, HIV-1 increases the
likelihood of successful infection following exposure at mucosal
surfaces. In future studies, it will be important to determine at
which point during sexual transmission the interaction between
HIV-1 and ?4?7is most relevant, and in addition whether targeting
this interaction can contribute to strategies designed to prevent
Cells and Reagents. Freshly isolated PBMCs were obtained from healthy donors
tor. (A) A Western blot stained with a an integrin ?7
polyclonal antisera of cell lysates derived from ?4?7high
CD4?T cells treated or not with the crosslinking re-
agent DTSSP followed by coprecipitation with protein
A agarose beads and the CD4 mAb OKT4 (lane 1),
IgG1?DTSSP (lane 2), and OKT4?DTSSP (lane 3). A
recombinant soluble ?4?7was run directly as a positive
control (lane 4). These results are representative of
three independent experiments. (B) A schematic de-
picting approximate sizes of ?4?7, CD4, and a gp120
Integrin ?7coprecipitates with the CD4 recep-
Cicala et al.PNAS ?
December 8, 2009 ?
vol. 106 ?
no. 49 ?
by negative selection using magnetic beads (StemCell Technologies). Cultured
CD4?T cells were activated with OKT3, IL2 (20 IU/mL) and RA (10 nM) unless
otherwise specified. Separation of ?4?7highvs. ?4?7low-negsubsets was carried out
using negative selection with mAbs CCR7 (150503) and CCR5 (2D7) followed by
magnetic selection with anti-mouse beads (Miltenyl). Gut-derived cells were
from each specified tissue/site and treated with collagenase (Sigma) using stan-
a study protocol approved by the Research Ethics Boards at the University of
Toronto and the Kenyatta National Hospital (Nairobi, Kenya). Retinoic acid was
obtained from Sigma Chemical. Integrin mAbs were purchased from Serotech,
Chemicon and R&D Systems, while other surface marker antibodies were pur-
was purchased from Lifespan Biosciences. Act-1 was provided by Dr. Stephen
used in conjunction with BD permeabilization buffer. Some mAbs were directly
recombinant envelope proteins were described in refs. 9 and 32. The full-length
chimeric proviral clone AD8-SF162 (33) was provided by Ronald L. Willey.
HIV Infection. AD8-SF162 was produced by transient transfection into 293T
fibroblasts. Viral stocks were normalized by p24 antigen ELISA (Perkin-Elmer).
washed and resuspended in fresh media containing RA and IL2. Viral replication
was measured by intracellular p24 staining (mAb RD1, Beckman Coulter) after
fixation and permeabilization using cytofix-cytoperm (BD Biosciences) or by p24
GAG antigen ELISA of culture supernatants.
Flow Cytometry Binding Assays. Cells were stained with fluorescein-labeled
with both mouse and human IgG. Staining buffer was freshly prepared and
MnCl2unless otherwise specified. Buffer without divalent cations contained 10
biotinylation reagent Pierce) followed by PE-conjugated neutravidin (Pierce).
Data were obtained using a BD FACSCalibur.
Confocal Microscopy. Freshly isolated gut-derived cells and RA-stimulated pe-
(eBioscience), and the CCR5 mAb 2D7 Alexa Fluor 488. Stained cells were fixed
laser lines. Fluorescence emission was collected as follows to minimize crosstalk:
415–465 nm pacific blue, 493–545 nm Alexa Fluor 488, and 575–620 nm Alexa
software (Bitplane AG).
CD4/?7CoPrecipitation. Purified RA/IL2 cultured CD4?T cells (20 ? 106) were
washed, resuspended in PBS and then treated for 30 min with 2 mM DTSSP
(Pierce) at 24 °C. The reaction was stopped with 20 mM Tris, pH 7.5, and lysed in
buffer containing 1% Triton X-100. Lysates were incubated with either OKT4 or
mouse IgG1 as specified, and precipitated with Protein G beads (GE Healthcare).
Precipitated proteins were electrophoresed on a 6% polyacrylamide gel under
reducing conditions and immunoblotted with an anti ?7polyclonal antisera
Data Analysis. Statistical analysis was performed with STATA/IC 10.0 (Stata
isons were performed using the non-parametric Wilcoxon signed-rank test for
paired sample. P ? 0.05 was considered significant.
Owen Schwartz, Juraj Kabat, and Lily Koo for assistance with imaging experi-
ments; Kathleen Kelly for sharing results ahead of publication; the National
was provided by the Intramural Research Program of the National Institutes of
coin. N Engl J Med 343:1020–1034.
2. Wagner N, et al. (1996) Critical role for beta7 integrins in formation of the gut-
associated lymphoid tissue. Nature 382:366–370.
3. Hawkins RA, Rank RG, Kelly KA (2000) Expression of mucosal homing receptor
genital mucosa in vivo. Infect Immun 68:5587–5594.
4. Kelly KA, Chan AM, Butch A, Darville T (2009) Two different homing pathways involving
following Chlamydia muridarum infection. Am J Reprod Immunol In press.
5. Haase AT (2005) Perils at mucosal front lines for HIV and SIV and their hosts. Nat Rev
acute and early human immunodeficiency virus type 1 infection. J Virol 81:599–612.
7. Brenchley JM, Price DA, Douek DC (2006) HIV disease: Fallout from a mucosal catas-
trophe? Nat Immunol 7:235–239.
8. Li Q, et al. (2005) Peak SIV replication in resting memory CD4? T cells depletes gut
lamina propria CD4? T cells. Nature 434:1148–1152.
9. Arthos J, et al. (2008) HIV-1 envelope protein binds to and signals through integrin
10. Wawer MJ, et al. (2005) Rates of HIV-1 transmission per coital act, by stage of HIV-1
infection, in Rakai, Uganda. J Infect Dis 191:1403–1409.
11. Derdeyn CA, et al. (2004) Envelope-constrained neutralization-sensitive HIV-1 after
heterosexual transmission. Science 303:2019–2022.
12. Mora JR, von Andrian UH (2004) Retinoic acid: An educational ‘‘vitamin elixir’’ for
gut-seeking T cells. Immunity 21:458–460.
14. Kelly KA, et al. (2009) The combination of the gastrointestinal integrin (alpha4beta7)
with Chlamydia trachomatis. Am J Reprod Immunol In press.
15. Hladik F, Hope TJ (2009) HIV infection of the genital mucosa in women. Curr HIV/AIDS
16. Vogel SS, et al. (2001) Exclusion of CD43 from the immunological synapse is mediated
by phosphorylation-regulated relocation of the cytoskeletal adaptor moesin. Immu-
17. Delon J, Kaibuchi K, Germain RN (2001) Exclusion of CD43 from the immunological
synapse is mediated by phosphorylation-regulated relocation of the cytoskeletal
Adaptor moesin. Immunity 15:691–701.
18. Kwong PD, et al. (2000) Structures of HIV-1 gp120 envelope glycoproteins from
laboratory-adapted and primary isolates. Structure 8:1329–1339.
are preferentially infected during acute SIV infection. Mucosal Immunol 2:439–449.
20. Krzysiek R, et al. (2001) Preferential and persistent depletion of CCR5? T-helper
lymphocytes with nonlymphoid homing potential despite early treatment of primary
HIV infection. Blood 98:3169–3171.
in heterosexual transmission of subtype A and C HIV-1. PLoS Pathog 5:e1000274.
22. Zhang ZQ, et al. (2004) Roles of substrate availability and infection of resting and
activated CD4? T cells in transmission and acute simian immunodeficiency virus
infection. Proc Natl Acad Sci USA 101:5640–5645.
4 beta 7 and LFA-1 in lymphocyte homing to Peyer’s patch-HEV in situ: The multistep
model confirmed and refined. Immunity 3:99–108.
24. Mehandru S, et al. (2004) Primary HIV-1 infection is associated with preferential
alpha4-integrin conformational activation. Biophys J 85:3951–3962.
26. Wu H, Kwong PD, Hendrickson WA (1997) Dimeric association and segmental variabil-
ity in the structure of human CD4. Nature 387:527–530.
of native HIV-1 gp120 trimers. Nature 455:109–113.
CD4? T cells by human immunodeficiency virus type 1. J Virol 79:13714–13724.
immunodeficiency virus type-1. Immunity 26:257–270.
2 and HIV in the female genital tract. AIDS 21:589–598.
of HIV. Cell 57:469–481.
33. Willey RL, Shibata R, Freed EO, Cho MW, Martin MA (1996) Differential glycosylation,
virion incorporation, and sensitivity to neutralizing antibodies of human immunode-
ficiency virus type 1 envelope produced from infected primary T-lymphocyte and
macrophage cultures. J Virol 70:6431–6436.
www.pnas.org?cgi?doi?10.1073?pnas.0911796106 Cicala et al.