Article

HIV-1-Infected CD4+ T Cells Facilitate Latent

Infection of Resting CD4+ T Cells through Cell-Cell
Contact
Graphical Abstract Authors
Luis M. Agosto, Melissa B. Herring,
Walther Mothes, Andrew J. Henderson

Correspondence
agosto@bu.edu (L.M.A.),
andrew.henderson@bmc.org (A.J.H.)

In Brief
Agosto et al. demonstrate that HIV-1 cell-
to-cell transmission between T cells
mediates the generation of latent
infection of resting CD4+ T cells. This
mechanism for the formation of latent
infection may be more difficult to reverse
and poses a potential hurdle for the
development of latency-reversing
therapies.

Highlights
d Developed in vitro model of HIV-1 latency incorporating HIV
cell-to-cell transmission

d HIV cell-to-cell transmission infects resting T cells without

robust T cell activation

d Proviruses generated are more difficult to induce, posing a

barrier to HIV eradication

Agosto et al., 2018, Cell Reports 24, 2088–2100

August 21, 2018 ª 2018 The Author(s).
https://doi.org/10.1016/j.celrep.2018.07.079

HIV-1-Infected CD4+ T Cells Facilitate Latent
Infection of Resting CD4+ T Cells
through Cell-Cell Contact
Luis M. Agosto,1,4,* Melissa B. Herring,1,2 Walther Mothes,3 and Andrew J. Henderson1,*
1Department of Medicine, Section of Infectious Diseases, Boston University Medical Center, Boston, MA, USA
2St. Georges University of London, London, UK
3Department of Microbial Pathogenesis, Yale University School of Medicine, New Haven, CT, USA
4Lead Contact
*Correspondence: agosto@bu.edu (L.M.A.), andrew.henderson@bmc.org (A.J.H.)
https://doi.org/10.1016/j.celrep.2018.07.079
SUMMARY
HIV-1 is transmitted between T cells through the
release of cell-free particles and through cell-cell
contact. Cell-to-cell transmission is more efficient
than cell-free virus transmission, mediates resis-
tance to immune responses, and facilitates the
spread of virus among T cells. However, whether
HIV cell-to-cell transmission influences the establish-
ment of HIV-1 latency has not been carefully
explored. We developed an HIV-1 latency model
based on the transmission of HIV-1 directly to resting
CD4+ T cells by cell-cell contact. This model recapit-
ulates the spread of HIV-1 in T-cell-dense anatomical
compartments. We demonstrate that productively
infected activated CD4+ T cells transmit HIV-1 to
resting CD4+ T cells in a cell-contact-dependent
manner. However, proviruses generated in this
fashion are more difficult to induce compared to pro-
viruses generated by cell-free infection, suggesting
that cell-to-cell transmission influences the estab-
lishment and maintenance of latent infection in
resting CD4+ T cells.
INTRODUCTION
HIV-1 spreads by both cell-free virions and by cell-contact-
dependent transmission between CD4+ T cells (Jolly et al.,
2004; Sattentau, 2008). The latter form of viral transmission is
several orders of magnitude more efficient than the spread of
cell-free particles (Chen et al., 2007; Zhong et al., 2013b). Two
important features may account for the high efficiency of cell-
to-cell viral transmission. First, cell contacts promote the forma-
tion of virally induced junctions, or virological synapses, between
CD4+ T cells, which concentrate large numbers of particles at
the site of cell-cell contact (Johnson and Huber, 2002; Jolly
et al., 2004; Phillips, 1994). This process delivers a high MOI to
the target cell (Del Portillo et al., 2011; Duncan et al., 2014; Zhong
et al., 2013a). Second, cell contact helps to overcome cellular
and environmental barriers that normally prevent infection by
2088 Cell Reports 24, 2088–2100, August 21, 2018 ª 2018 The Author(s).
This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Cell Reports
Article
cell-free HIV, such as antiviral factors, neutralizing antibodies,
some anti-retroviral therapies, and poor viral fitness (Agosto
et al., 2015b; Brandenberg et al., 2014; Gupta et al., 1989; Jolly
et al., 2010; Mathez et al., 1993; Sigal et al., 2011; Zhong et al.,
2013a). However, despite the efficiency of cell-to-cell HIV-1
transmission, it remains unclear whether this process contrib-
utes to the pathogenesis of HIV.
The most significant barrier for the eradication of HIV-1 infec-
tion is the existence of a small pool of latently infected cells that
survive despite long-term suppression of viral replication by
highly active anti-retroviral therapy (HAART) (Finzi et al., 1997;
Ruelas and Greene, 2013; Strain et al., 2003). Following treat-
ment interruption, this pool of cells contributes to the re-emer-
gence of viremia (Chun et al., 1997b; Davey et al., 1999; Wong
et al., 1997). The reservoir of latently infected T cells is estab-
lished within 3–10 days after exposure to virus and prior to the
detection of viremia (Chun et al., 1998; Whitney et al., 2014).
The vast majority of these latently infected cells are composed
of resting memory CD4+ T cells that have a long half-life and
are maintained by homeostatic proliferation without the induc-
tion of viral replication (Brenchley et al., 2004; Chomont et al.,
2009; Chun et al., 1997a; Douek et al., 2002; Maldarelli et al.,
2014; Ostrowski et al., 1999; Wagner et al., 2014). Significant
progress has been made toward understanding the molecular
mechanisms that regulate proviral transcription and latency in
T cells. Latently infected T cells are characterized by inefficient
proviral transcription due to insufficient expression and binding
of cellular transcription factors to the proviral promoter, the inef-
ficient elongation of transcription, and a closed chromatin envi-
ronment (Agosto et al., 2015a; Mbonye and Karn, 2017; Ruelas
and Greene, 2013). Despite the knowledge of these molecular
mechanisms that regulate latent infection, it remains unclear
how the pool of latently infected resting CD4+ T cells is initially
generated.
Early studies suggested that cell activation, as defined by cell
cycle progression beyond G1b, is not only required for proviral
production but also for efficient reverse transcription and pro-
viral integration (Bukrinsky et al., 1991; Korin and Zack, 1998;
Stevenson et al., 1990; Sun and Clark, 1999; Unutmaz et al.,
1999; Zack et al., 1992). This led to the hypothesis that latently
infected cells are generated as activated HIV-infected CD4+
T cells return to a resting state (Ruelas and Greene, 2013).
 Wash Alu-PCR Targets
+SQV

48-72h aCD4 24h 72h

aCD4 rCD4
FACS for HIV+
+RAL Targets
Co-Culture +Stimulation
HIV 1 donor: 1 target 24h rCD4

1K +EFV No Drug 100

800 80

Frequency (%)
0.5% 23.3+/-2.6%

Count
FSC

600 60
400 40
200 20
0 0 1 2 3 4 0
10 10 10 10 10 100 101 102 10
3
10
4
0 102 103 104 105 Targets Viability
HIV-Gag CD69/HLA-DR/CD25
Resting cells
Activated cells
Unstained

Figure 1. In Vitro Model of HIV-1 Cell-to-Cell Transmission

See text and Experimental Procedures for details. A flow cytometry profile for HIV Gag expression in activated CD4+ T cells in the presence or absence of EFV is
shown. The average percent of HIV-1 Gag expression ± SD from 8 experiments is indicated. A flow cytometry profile of activation marker expression among
purified resting CD4+ T cells and activated CD4+ T cells is shown. We estimated the percent of target resting CD4+ T cells and their viability at the end of the co-
culture period.

An alternative mechanism for the formation of latently infected
CD4+ T cells is that resting cells are directly infected. Although
direct infection of resting CD4+ T cells has been estimated to
be 10-fold less efficient compared to activated cells, the pro-
cess can be successfully completed but with slower kinetics
than in activated cells (Agosto et al., 2007; Lassen et al.,
2012; Plesa et al., 2007; Swiggard et al., 2005; Vatakis et al.,
2007). Resting CD4+ T cells represent a large proportion of
the total population of CD4+ T cells, and because these cells
are already at a cellular state that would favor latent infection,
it is possible that directly infected resting cells may represent
a significant proportion of the latent reservoir. Understanding
the requirements for infection of these cells is therefore impor-
tant for better understanding HIV-1 pathogenesis (Zack et al.,
2013). Several factors could influence the susceptibility of
resting CD4+ T cells to HIV-1 infection without resulting in full
activation of the cell and progression through the cell cycle.
These factors may include the cytokine environment in tissues
and cell-cell interactions (Eckstein et al., 2001; Kinter et al.,
2003; Nishimura et al., 2005; Saleh et al., 2007; Shen et al.,
2013; Zhang et al., 1999). Indeed, as previously shown by
Evans et al. (2013) and Kumar et al. (2015), cell-cell interac-
tions, such as those between antigen-presenting cells and
CD4+ T cells, can mediate latent infection in CD4+ T cells.
Thus, it is likely that cell-to-cell transmission between CD4+
T cells may facilitate the establishment of latency by increasing
the likelihood of direct infection of resting CD4+ T cells.
In the present study, we developed a model of HIV-1 latency
that incorporates HIV-1 cell-to-cell transmission directly to
resting CD4+ T cells. With this model, we demonstrate that
productively infected CD4+ T cells mediate latent infection
in target resting CD4+ T cells in a cell-contact-mediated
fashion. Infection via this mechanism generates a population
of cells harboring latent proviruses that are more difficult to
reactivate. Taken together, our studies support a mechanism
for the generation of latently infected cells through direct infec-
tion of resting CD4+ T cells and provide evidence for the
importance of T cell-T cell interactions in the pathogenesis
of HIV.
RESULTS
Modeling HIV-1 Cell-to-Cell Transmission to Resting
CD4+ T Cells
To test whether productively infected activated CD4+ T cells
transmit HIV-1 to resting CD4+ T cells, we employed a co-cul-
ture approach in which activated CD4+ T cells productively in-
fected with HIV-1 were co-cultured with autologous target
resting CD4+ T cells (Figure 1). 20%–30% of infected activated
cells expressed HIV-1 as assessed by intracellular HIV-1 Gag
(Figure 1). Resting CD4+ T cells were purified from total CD4+
T cells by depleting cells expressing CD25, CD69, and HLA-
DR (Figure 1); absence of these markers corresponds to
T cells at the quiescent and/or resting G0/G1a stage of the cell

Cell Reports 24, 2088–2100, August 21, 2018 2089

cycle (Agosto et al., 2009; Korin and Zack, 1998). Resting CD4+
T cells were stained with eFluor670 prior to co-culture to allow
us to track them at the end of the incubation period. The cells
were co-cultured for 24 hr and were then treated with the HIV
protease inhibitor saquinavir (SQV). Treatment with SQV pre-
vents maturation of viral particles released from infected acti-
vated CD4+ T cells, thus preventing the spread of de-novo-
assembled virus. This treatment allowed us to examine the
transfer of HIV-1 that occurs specifically within the first 24 hr
of co-culture (Agosto et al., 2014; Titanji et al., 2013). Once
SQV was added, the culture was incubated for 3 days to allow
time for HIV-1 to reverse transcribe and integrate in the target
cells. This extended incubation is necessary as the kinetics of
reverse transcription and integration are slower in resting cells
(Plesa et al., 2007). At the end of the co-culture period, we
confirmed that the percent of target resting CD4+ T cells was
maintained at approximately 50% and that their viability was
maintained at approximately 80% (Figure 1). At this point, target
cells were separated from infected activated cells by cell sorting
and assayed for proviral DNA by Alu-PCR (Figure 2). This PCR
approach, previously described in detail (Liszewski et al.,
2009), is based on an initial amplification of integrated HIV using
a reverse primer specific for the HIV gag gene and a forward
primer specific for human Alu. A second quantitative nested
PCR amplification is performed with the reaction product of
the first amplification as a template, using HIV-specific primers
recognizing the R-U5 region and a TaqMan probe (Figure 2B).
Signals from experimental samples were plotted against a poly-
clonal integration standard to determine the number of proviral
copies per cell. This Alu-PCR technique has a detection sensi-
tivity of a single HIV integration event per 10,000 cells (Agosto
et al., 2007). Treatment of cultures with the anti-retroviral drug
efavirenz (EFV) provides the background signal for both qPCR-
and flow cytometry-based experiments. We detected proviral
DNA in target resting CD4+ T cells after co-culture with produc-
tively infected activated CD4+ T cells compared to co-cultures
treated with EFV (Figure 2C). The amount of proviral DNA corre-
sponded to an infection rate of 1%–15% of resting CD4+ T cells.
Importantly, we detected integration in target resting CD4+
T cells when using both X4-tropic HIV-1 (HIVNL4-3) and the
more physiologically relevant R5-tropic HIV-1 (HIVREJO). This is
consistent with expression of CXCR4 and CCR5 expression
on resting CD4+ T cells (Figure S1; Dai et al., 2009; Tabler
et al., 2014; Zerbato et al., 2016).
Cell-cell interactions and the culture microenvironment can
affect the phenotype and function of cells. We wanted to deter-
mine whether the co-culture microenvironment leads to cell
activation of the resting CD4+ T cells. We monitored the expres-
sion of the activation markers CD69, HLA-DR, and CD25 among
the target resting CD4+ T cell population at various time points
post-co-culture by flow cytometry. We observed that, although
target resting CD4+ T cells did not demonstrate an activated
phenotype throughout the co-culture when compared to acti-
vated CD4+ T cells, there was a modest but statistically signif-
icant induction of T cell activation marker expression (Figure 3).
These data suggest that weak cell signaling is involved in the
process of HIV-1 cell-to-cell transmission to target resting
CD4+ T cells.
2090 Cell Reports 24, 2088–2100, August 21, 2018
Productively Infected Activated CD4+ T Cells Form
Contacts with Resting CD4+ T Cells
We examined whether infection of target resting CD4+ T cells
following co-culture with productively infected activated
CD4+ T cells was dependent on cell-cell contact. To test this,
resting CD4+ T cells and productively infected activated
CD4+ T cells were co-cultured as described above or sepa-
rated by a 0.4-mm membrane Transwell (Figure 4A). We then
assessed the level of HIVNL4-3 integration by Alu-PCR after
72 hr of culture. Whereas HIV integration is detected in resting
cells after co-culture, we were unable to detect proviral DNA
PCR signal in Transwell cultures above the background signal
from EFV-treated cultures (Figure 4B). To confirm that viral par-
ticles released by productively infected CD4+ T cells cross the
0.4-mm membrane, we plated the HIV indicator cell line TZMbl
in the lower well. This indicator cell line, which is highly suscep-
tible to HIV-1, is transduced with an HIV Tat-responsive re-
porter construct that expresses firefly luciferase upon HIV
infection. We detected a significant luciferase signal above
background, confirming that viral particles crossed the mem-
brane and successfully infected cells in the bottom well (Fig-
ure 4C). Taken together, these data suggest that T cell-T cell
contact more efficiently facilitates the transfer of HIV-1 from
productively infected cells to resting CD4+ T cells compared
to the release of cell-free particles in our co-culture system.
This observation is consistent with published work conducted
with activated CD4+ T cells and cell lines indicating that cell-
cell-contact-mediated HIV-1 transmission is largely responsible
for the spread of HIV in culture (Chen et al., 2007; Sourisseau
et al., 2007; Zhong et al., 2013a).
To directly assess whether productively infected CD4+ T cells
interact with resting CD4+ T cells and to gain insight as to what
modes of viral transmission are involved, we visualized cell-cell
contacts by confocal microscopy. We infected activated CD4+
T cells with HIVNL4-3-Gag-GFP and co-cultured them with resting
CD4+ T cells for 2 hr prior to confocal microscopy (Figure 4D).
Because HIVNL4-3-Gag-GFP particles are fluorescent, we deter-
mined the frequency of HIV-1 Gag-GFP fluorescence associated
with target cells in cell-cell contacts (Figure 4E) compared to the
frequency of fluorescence in resting CD4+ T cells that were not
engaged in cell-cell contacts (Figure 4F). We observed that
target resting CD4+ T cells were more likely associated with
HIV-1 Gag-GFP fluorescence when conjugated with activated
CD4+ T cells compared to cells not in contact with activated
CD4+ T cells (Figure 4G).
Formation of cell-cell contacts is a dynamic process often re-
sulting in the polarization of HIV assembly to the sites of cell-cell
contact. This polarization of viral assembly is a defining charac-
teristic of virological synapses (Chen et al., 2007; Jolly et al.,
2004). Therefore, we monitored the formation of cell-cell conju-
gates using live-cell imaging, quantified the duration of the con-
jugates, and surveyed the frequency of HIV Gag-GFP polariza-
tion to the site of cell-cell contact (Figure 5; Videos S1, S2, and
S3). We observed that productively infected activated CD4+
T cells formed conjugates with resting CD4+ T cells and could
observe the transfer of particles in some instances, although
we lack the resolution to see the transfer in all cell-cell conju-
gates. Approximately 26% of conjugates resulted in the
 A

B C

Figure 2. HIV-Producing T Cells Transmit HIV-1 Directly to Resting CD4+ T Cells

(A) After co-culture, target resting CD4+ T cells were sorted to assess the level of HIV-1 integration within this population. The flow cytometry plots show the
sorting strategy.
(B) Simplified diagram of the Alu-PCR strategy to specifically detect HIV integration. Shown are the relative primer locations.
(C) HIV-1 integration was assessed in co-cultures infected with HIVNL4-3 (X4) and HIVREJO (R5) by Alu-PCR. Parallel control co-cultures were treated with EFV.
Symbols represent independent blood donors. Integration was measured 5 times for each sample. **p < 0.01.
Error bars represent the SD of the mean.

accumulation of HIV Gag at the site of cell-cell contact. Conju- vations support our hypothesis that cell-cell contacts facilitate
gates that resulted in the polarization of HIV Gag-GFP were the transfer of HIV-1 to resting CD4+ T cells in co-cultures.
generally longer lived (average 33 min) compared to conjugates Several modes of cell-contact-dependent viral transmission
that did not result in polarization (average 23 min). These obser- between cells have been described. In the case of HIV-1, virus

Cell Reports 24, 2088–2100, August 21, 2018 2091

A B We detected a significant amount of HIV-1 Gag expression in

CD25/CD69/HLA-DR (MFI)
10 4
target cells after stimulation of the co-culture compared to unsti-
mulated co-cultures (Figure 7A). The presence of inducible pro-
10 3 viral HIV-1 expression supports the conclusion that co-culture
Count

* * * with productively infected activated CD4+ T cells leads to the

2
10 generation of latent infection in resting CD4+ T cells.
The ratio of the percent HIV-1 Gag-positive cells to the percent
10 1 cells with integrated HIV-1 provides insight into the subset of
2 3 4 5
0 10 10 10 10
cells that have reversible latent infection. For the example shown
D ore e
C

D y1 e
2 CC

D y2 e
C
ay ne

ed
iv C
ef p

D lon
C

A 4C
B ty

at
CD69/HLA-DR/CD25 l

l
o

A
in Figure 7A, approximately 1.07% of the target resting CD4+
Is

ct
1

4
a

a
D

D
ay

ay

ay
Isotype Day 2
Day 0
Day 4
Day 1 Activated
Figure 3. Co-culture with Productively Infected Activated CD4+
T Cells Does Not Induce Robust Activation of Resting CD4+ T Cells
(A) We assessed the level of T cell activation among target resting CD4+ T cells
at various time points post-co-culture based on the expression of CD69, HLA-
DR, and CD25. The levels of activation based on the expression of activation
markers (mean fluorescence index) were compared to activated CD4+ T cells
treated with PHA, IL-2, and IL-7.
(B) Combined data from 3 individual experiments from three different blood
donors. Cells cultured alone or in co-culture (CC) with infected CD4+ T cells are
shown. The horizontal line represents the average of each population. *p <
0.05.
cell-to-cell transmission can be mediated by virological synap-
ses as previously stated. However, HIV-1 particles can also be
transmitted without polarization. For example, HIV-1 particles
produced by infected cells can be retained by infected cells
and transferred to adjacent uninfected cells (Sherer et al.,
2010; Zhong et al., 2013b) or particles can be captured by unin-
fected cells and subsequently transferred to other uninfected
cells. We examined our co-cultures using microscopy to deter-
mine whether there was evidence of alternative modes of viral
transmission. We detected both cell-cell contacts with polarized
HIV-1 assembly (Figure 6A) and cell contacts without polarized
virus (homogeneous cytoplasmic GFP staining and CD4
depleted; Figure 6B). We also found cell contacts between unin-
fected cells carrying viral particles and uninfected resting CD4+
T cells (GFP punctae and CD4-positive; Figure 6C). This obser-
vation suggests that uninfected T cells carrying bound HIV could
contribute to the infection of resting CD4+ T cells. We observed
that the frequency of GFP signal on resting CD4+ T cells in the
context of polarized HIV was approximately 40%. Whereas
these images suggest that both polarized and non-polarized viral
transmission lead to the transfer of viral particles to resting CD4+
T cells, single snapshots may not fully capture the dynamic pro-
cess of viral transmission (Figure 6D).
Latent Infection in the Target Cell Population Is
Inducible
Because we could detect HIV-1 integration among target resting
CD4+ T cells after transmission from productively infected
T cells, we tested whether proviral expression was inducible.
As described in Figure 1, co-cultures were treated with raltegra-
vir and stimulated with a combination of anti-CD3/CD28 beads,
interleukin-2 (IL-2), and IL-7. HIV-1NL4-3 expression was as-
sessed by intracellular Gag staining and flow cytometry analysis.
T cells express HIV-1 Gag once the percent background expres-
sion from unstimulated target cells is subtracted. This percent-
age reflects the proportion of cells with inducible proviruses.
Before stimulation, we estimated that 4.4% of the total target
resting CD4+ T cell population (or 0.044 proviruses/cell) carried
integrated HIV-1 as determined by Alu-PCR. Therefore, we
calculated, by dividing the percent of HIV-Gag-positive cells by
the percent of cells carrying proviruses, that approximately
24% of infected cells could be induced to produce HIV-1 upon
stimulation. Based on four independent experiments, an average
of 2.6% of resting cells carried proviruses after co-culture infec-
tion (Figure 7D) and an average of 21% of proviruses were induc-
ible (Figure 7E). To gain further insight into the relative efficiency
of the induction of proviruses in resting CD4+ T cells after cell-to-
cell transmission, we tested the induction of latent infection
generated by spinoculating resting CD4+ T cells with HIVNL4-3.
This model of HIV-1 latency involves the centrifugation of
concentrated HIV-1 supernatant directly onto resting CD4+
T cells (Swiggard et al., 2005). Because pure resting CD4+
T cells are relatively resistant to HIV-1 infection compared to acti-
vated T cells, delivering a larger amount of viral particles in-
creases the proportion of resting cells that become infected
while preserving the resting nature of the cells (Agosto et al.,
2007, 2009). Differences in the induction efficiency of proviruses
between the co-culture model and the spinoculation model
would suggest that co-culture conditions could have a direct
impact on the nature of latent infection in target resting CD4+
T cells. We infected resting CD4+ T cells from 4 blood donors
by spinoculation and obtained HIV-1 expression in all donors
upon induction with a combination of anti-CD3/CD28 beads,
IL-2, and IL-7 in the presence of raltegravir (Figure 7B). In the
example shown in Figure 7B, 2.87% of cells expressed HIVNL4-3
Gag (background from unstimulated cells subtracted), and about
6.2% of the resting CD4+ T cells carried integrated HIV-1 (or
0.062 proviruses/cell) prior to stimulation. Therefore, we found
that approximately 46% of proviruses were induced upon stim-
ulation of the cells. From 5 experiments, an average of 13.5%
of resting cells carried proviruses after spinoculation (Figure 7D)
and an average of 43% of proviruses were inducible (Figure 7E).
The fraction of inducible proviruses after spinoculation
compared to co-culturing is statistically higher and suggests
that co-culture conditions contribute to the generation of provi-
ruses more resistant to induction.
To examine other more physiological modes of infection of
resting CD4+ T cells, we also investigated the proportion of
inducible proviruses when resting CD4+ T cells were infected
with concentrated HIVNL4-3 without spinoculation. To achieve
detectable HIV infection in resting CD4+ T cells without

2092 Cell Reports 24, 2088–2100, August 21, 2018

 A B NS
C
10 0 1500 *

Infectivity (Luc RLU)

**

Proviruses/cell
-1
10
1000
10 -2
10 -3
500
10 -4
Co-culture Cell-free
10 -5 0
HIV-1 donor cells +EFV No Drug

re

l
FV

el
Target cells

tu

sw
+E

ul

an
C
o-

Tr
C
gag tat

D MA CA NC p6 gfp vif vpu

U3 R U5 U3 R U5
Δpol vpr env nef

rev

E Cell-Cell Contact F No Cell-Cell Contact G

rCD4T w/ GFP Signal (%)

100
*
80
60
P
40
20
0
T No Contact
Contact

Figure 4. HIV-1 Integrates in Resting CD4+ T Cells in a Cell-Contact-Dependent Manner

(A) A Transwell insert system was used to compare the relative efficiency of resting CD4+ T cell infection after co-culture with HIV-1-infected activated cells or
when the infected activated cells were separated.
(B) HIV-1 integration was measured by Alu-PCR in the target resting CD4+ T cells after co-culture or Transwell-based infection. Note that the co-culture dataset is
the same as Figure 1B for HIVNL4-3.
(C) To confirm that viral particles produced by infected activated CD4+ T cells can diffuse through the membrane, TZMbl cells were plated in the bottom well.
Infection was assessed by luciferase expression. Error bars represent the SD of 3 measurements of luciferase. A parallel control culture was treated with EFV.
(D) Diagram of the HIVNL4-3-Gag-GFP genome.
(E and F) Activated CD4+ T cells producing HIVNL4-3-Gag-GFP (green, P) were co-cultured with target resting CD4+ T cells pre-labeled with CMAC (blue, T) and
stained for CD4 (red). An example of a resting CD4+ T cell that carried HIV-1 Gag-GFP signal and was also in contact with an HIV-1-producer T cell is shown in (E).
An example of a resting CD4+ T cell that was not in contact with HIV-1-producer T cells but carried HIV-1 Gag-GFP signals is shown in (F). The scale bars in the
images represent 5 mm.
(G) The relative proportion of each is shown.
Error bars represent the SD of the mean. In (B), (C), and (G), *p < 0.05, **p < 0.01.

spinoculation, we used 5 times concentrated HIVNL4-3 compared
to the spinoculation experiments. Under these conditions, we
observed inducible latent HIV in 3 out of 5 experiments (Fig-
ure 7C). From these experiments, we detected an average of
5.6% of resting cells carrying proviruses (0.056 proviruses/cell;
Figure 7D). Importantly, this level of HIV-1 integration is compa-
rable to the levels detected in co-culture experiments. On
average, 31% of proviruses were inducible (Figure 7E). This pro-
portion of inducible proviruses is intermediate between that of
proviruses generated by cell-to-cell transmission and proviruses
generated by spinoculation. The observed differences in the abil-
ity to reactivate provirus in different models of infection of resting
cells suggests that reversing latent proviruses partly reflects how
the infection was initially established.
DISCUSSION
Cell-contact-mediated spread of HIV-1 has been estimated to
be at least an order of magnitude more efficient compared to
passive dissemination of particles through the extracellular
milieu. Thus, this form of viral transmission can have significant
effects on the pathogenesis of HIV. First, this mode of transmis-
sion can mediate the spread of HIV-1 within T-cell-dense
anatomical compartments (Law et al., 2016; Murooka et al.,

Cell Reports 24, 2088–2100, August 21, 2018 2093

A Figure 5. Contacts between Productively In-
B this mechanism, several cell line and
**
60
Time (min)
40
20
0
No Polar. Polarized
2012; Sewald et al., 2012, 2015). Second, the ability of cell-to-
cell transmission to generate infected cells with multiple provi-
ruses could facilitate recombination, thus increasing viral diver-
sity (Dang et al., 2004; Del Portillo et al., 2011; Dixit and Perelson,
2004; Jung et al., 2002). Third, HIV-1 cell-to-cell transmission
could be an important mechanism for the massive depletion of
CD4+ T cells in untreated infected patients (Doitsh et al.,
2010). Our present study proposes that an additional role of
HIV-1 cell-to-cell transmission in vivo is the generation of latently
infected cells by direct infection of resting CD4+ T cells.
Latently infected resting CD4+ T cells represent the most
important source of rebounding virus when treatment is interrup-
ted (Ruelas and Greene, 2013). The pool of latently infected cells
is comprised of a phenotypically heterogeneous population of
cells (Churchill et al., 2016). Therefore, a deep understanding
of how these cells are generated and maintained and how pro-
viral latency is regulated is essential for the development of
2094 Cell Reports 24, 2088–2100, August 21, 2018
fected CD4+ T Cells and Uninfected Resting
CD4+ T Cells Result in Polarization of HIV
Gag to the Site of Cell-Cell Contact
(A) Live-cell imaging was conducted to observe
conjugate formation between activated CD4+
T cells infected with HIVNL4-3-Gag-GFP (green) and
uninfected resting CD4+ T cells in real time. Images
were collected every 2.5 min. Shown are selected
time points when the transfer of particles was de-
tected (white arrows) and when polarization of HIV
Gag-GFP to the site of cell-cell contact was
detected (orange arrow). The full movie is provided
as Video S1. The scale bars in the images repre-
sent 5 mm.
(B) Cell-cell contacts were identified and scored
based on their duration. Contacts maintained for
least 5 min were scored. The duration of cell-cell
contacts that did not result in HIV Gag-GFP polari-
zation were compared to the duration of contacts
that resulted in polarization. Data shown represent
all cell-cell contacts identified in 6 experiments from
2 blood donors. The horizontal line represents the
average of each population. **p < 0.01.
any curative approach. The most widely
accepted mechanism for the generation
of latently infected resting CD4+ T cells is
the return of fully or intermediately acti-
vated cells to a resting state after infection
with HIV-1 (Shan et al., 2017). Based on
primary cell in vitro models have been
developed over the years to study HIV-1
transcription and latency (Pace et al.,
2011). These models have been instru-
mental in the characterization of funda-
mental processes that regulate HIV-1
transcription and latency. However, these
models are limited by the use of cells that
may not fully represent the natural pool of
latently infected cells in vivo and may be
biased toward chromatin-dependent repression. Models of
HIV-1 latency based on direct infection of resting CD4+ T cells
have been less popular, partly because of the relative resistance
of these cells to HIV-1 infection. These cells have been shown to
express some restriction factors that limit HIV infection (Baldauf
et al., 2012; Zack et al., 2013). Despite the presence of these fac-
tors, it has been demonstrated that resting cells can still be in-
fected with HIV directly and at a sufficient frequency to allow
the study of HIV latency in this context (Agosto et al., 2007; Las-
sen et al., 2012; Plesa et al., 2007; Swiggard et al., 2005; Vatakis
et al., 2007). Studying HIV latency in the context of direct infec-
tion of resting CD4+ T cells can overcome some of the shortcom-
ings of models involving activated T cells and cell lines. In vitro
models of direct infection of primary resting CD4+ T cells pre-
serve the natural diversity of cellular phenotypes because artifi-
cial manipulations are minimized. Resting CD4+ T cells are
already at a state predisposed to proviral latency; hence,
 A HIV Gag-GFP CD4-Alexa647 Merged

P T P T P T

B C D
NS

rCD4T w/ GFP Signal (%)

100
P
T
80

T 60
T
40
T
P
20
0
P Polarized No Polar.

Figure 6. Visualization of Viral Cell-to-Cell Transmission to Resting CD4+ T Cells

(A) Example of polarized HIV-1 assembly as defined by the accumulation of HIV-1 Gag in HIV-1-producing activated cells (P) and CD4 in target cells (T) at the site
of cell-cell contact (arrow).
(B) Example of resting CD4+ T cells carrying particles while in contact with productively infected T cells without polarization of viral assembly.
(C) Example of a resting CD4+ T cell carrying viral particles while in contact with uninfected activated T cells also carrying particles.
(D) The proportion of resting CD4+ T cells that acquired Gag-GFP from polarized viral assembly versus non-polarized viral assembly was quantified by counting
cell-cell contacts.
The scale bars in the images represent 5 mm. Error bars represent the SD of the mean.

studying HIV latency in this context could reveal previously un-
recognized mechanisms for regulating HIV transcription. The
in vitro model presented in this study has the added benefit of
incorporating T cell-T cell interactions as part of the process of
generating latent infection. With this model of HIV-1 latency,
we demonstrate that productively infected activated CD4+
T cells directly transmit HIV-1 to uninfected resting CD4+
T cells, leading to the generation of pools of latently infected
resting CD4+ T cells. This mechanism for the generation of
latently infected cells has been underappreciated and likely rep-
resents an important outcome of HIV-1 spread in vivo before the
initiation of effective therapy.
Initial evidence that cell-contact-mediated transmission of
HIV-1 is relevant for the generation of latently infected cells
was suggested in the context of transmission from dendritic cells
to resting CD4+ T cells (Evans et al., 2013; Kumar et al., 2015). As
dendritic cells probe for antigens, they capture and retain full
particles largely in a CD169-dependent fashion (Izquierdo-
Useros et al., 2012; Puryear et al., 2013; Sewald et al., 2015).
As they interact with resting CD4+ T cells during antigen presen-
tation, live particles are transmitted to target cells (McDonald,
2010). In addition, cell-cell contact and the tissue microenviron-
ment also help overcome cell resistance to infection and influ-
ence the regulation of target cell infection (Shen et al., 2013).
Our study explores infection of resting CD4+ cells by cell-to-
cell transmission between T cells. These interactions are funda-
mentally different than interactions involving dendritic cells or
endothelial cells. T cells typically do not engage in direct antigen
presentation with each other; thus, signaling through the T cell
receptors and co-stimulatory receptors is unlikely to be antigen
specific (Len et al., 2017). Therefore, the set of signals involved in
the process of transmitting HIV-1 to resting cells in the context of
T cell-T cell interactions is likely unique. It is possible that the
specific signals and environment created during the transmis-
sion of HIV-1 to resting CD4+ T cells from activated T cells will
affect the establishment of inducible latent infection. This lack
of robust T cell signaling, as in the context of antigen presenta-
tion, likely prevents productive infection and directs proviruses
toward a latent state. Our results suggest that proviruses gener-
ated by cell-cell contact between T cells may be harder to induce
compared to proviruses generated by cell-free infection. This
could be a reflection of the route through which latent infection
was generated, the phenotype of cells that harbor latent provi-
ruses, differences in the proportion of defective proviruses, or

Cell Reports 24, 2088–2100, August 21, 2018 2095

A Co-Culture Figure 7. HIV-1 Latent Infection Generated by
Cell-to-Cell Transmission Is Less Inducible

HIV Expression (% Gag+)

Unstimulated Stimulated
3 Than Latent Infection Generated by Cell-
free HIV
(A) Co-cultures of productively infected activated
2
* CD4+ T cells with resting CD4+ T cells were stimulated
FSC-A

with CD3/CD28 beads, IL-2, and IL-7 in the presence

of raltegravir. At 24 hr post-stimulation, HIV-1
1 expression among target cells was assessed by
intracellular HIV-1 Gag staining and flow cytometry
0.651% 1.72% analysis. A representative plot is shown comparing
2 3 4 5 2 3 4 5
0 unstimulated with stimulated cells. A dot plot of the
0 10 10 10 10 0 10 10 10 10 percent of target T cells expressing HIV Gag after

im

28
St

3/
HIV-Gag stimulation or without stimulation is shown. Each

D
ot

+C
symbol represents an independent experiment with

N
Treatment different blood donors. The horizontal line represents
B Cell-Free Spinoculation the average of each population.
(B) Resting CD4+ T cells were spinoculated with
HIV Expression (% Gag+)
Unstimulated Stimulated
15 HIVNL4-3 and incubated for 3 days. At this point, the
** cells were stimulated and analyzed for HIV-1 expres-
sion as in (A). A representative plot is shown
10 comparing unstimulated with stimulated cells.
FSC-A

(C) Resting CD4+ T cells were infected with HIV-1

without spinoculation and incubated for 3 days. At this
1.51% 4.38% 5 point, the cells were stimulated and analyzed for HIV-1
expression as in (A). A representative plot is shown
comparing unstimulated with stimulated cells.
2 3 4 5
0 (D) HIV integration in target resting CD4+ T cells was
2 3 4 5
0 10 10 10 10 0 10 10 10 10 measured under co-culture, spinoculation, and no-
im

28
St

spinoculation infection conditions. Background PCR

3/

HIV-Gag
D
ot

signal was determined based on samples treated

+C
N

Treatment with EFV.

C Cell-Free (E) The proportion of inducible proviruses was calcu-
Unstimulated Stimulated lated from the percent of HIV-positive cells based on
NS
HIV Expression (% Gag+)

flow cytometry after stimulation and after subtracting

3
the background signal from unstimulated cells divided
by the percent HIV-positive cells based on Alu-PCR.
2 This fraction is plotted for 4 independent experiments
FSC-A

0.815% 2.67% done under co-culture conditions and 5 independent

experiments for spinoculation and non-spinoculation
1 infection conditions.
Error bar represent the SD of the mean. In (A), (B), (D),
and (E), *p < 0.05, **p < 0.01.

0 10
2 3
10 10
4
10
5 2
0 10 10
3 4
10
5
10 0
im

28

HIV-Gag
3/
St

D
ot

+C
N

Treatment
D E NS
10 0 0.8
** * *
Ratio Gag+/Provirus

*
(proviruses/cell)
HIV Integration

10 -1 0.6

10 -2 0.4

10 -3 0.2

10 -4 0.0
e

in
C V

el V

e
FV

in
Sp tur

r
re

re
F

F
Sp

Sp
tu
+E

+E
+E

l-F

l-F
ul

ul

F-

el
C
C

in

C
C
o-

o-
C

C
C

Infection Type Infection Type

2096 Cell Reports 24, 2088–2100, August 21, 2018

the presence of multiple proviruses in a small number of in-
fected cells (Chavez et al., 2015; Maldarelli, 2016; Rezaei
et al., 2018; Tsunetsugu-Yokota et al., 2016). Nevertheless,
our initial observation that latent infection generated by cell-
to-cell transmission is not easily reversible requires further
investigation, as current approaches to purging the latent reser-
voir may not be effective in this context. Our proposed model
could be optimized for testing this possibility and could help
in the search for novel compounds that are more efficient at
purging latent provirus.
EXPERIMENTAL PROCEDURES
Cells
Peripheral blood mononuclear cells were purified from leukapheresis packs
using the Lymphoprep gradient (StemCell Technologies). CD4+ T cells were
negatively selected using the EasySep Human CD4+ T Cell Enrichment Kit
(StemCell Technologies). A fraction of purified CD4+ T cells was stimulated
with phytohaemagglutinin (PHA-P) (Sigma-Aldrich), IL-2, and IL-7 prior to
infection. Another fraction of cells was preserved in medium without exoge-
nous cytokines until needed and then purified the resting cell fraction for co-
culture experiments by depleting cells expressing CD25, CD69, and human
leukocyte antigen (HLA)-DR. TZMbl cells were obtained from the NIH AIDS Re-
agents Program.
Viruses
Viruses used for infections of CD4+ T cells were generated by transfection of
HEK293T cells with plasmids encoding the HIV-1 clones NL4-3 or REJO.c
(AIDS Reagents Program). Viral supernatants were concentrated by ultra-
centrifugation. Concentrated stocks were subsequently titered in CEM-GFP
cells (AIDS Reagents Program) engineered to express human CCR5.
Co-culture Experiments
Activated CD4+ T cells were infected with HIV-1 by spinoculation and incu-
bated for 48–72 hr at 37 C in the presence of IL-2 and IL-7. Before co-culture,
infected cells were washed to remove cytokines and unbound viral particles.
Infected cells were then co-cultured with target resting CD4+ T cells. Parallel
control co-cultures were treated with EFV (AIDS Reagents Program). The
co-cultures were incubated for 24 hr, treated with SQV (AIDS Reagents Pro-
gram), and incubated for an additional 3 days. At this point, some co-cultures
were treated with raltegravir (AIDS Reagents Program) to block further HIV-1
integration and were stimulated with anti-CD3/CD28 beads (Invitrogen) in
the presence of IL-2 and IL-7 for 24 hr prior to flow cytometry analysis of
HIV-1 expression. Other co-cultures were not stimulated but were prepared
for sorting of target cells and subsequent PCR analysis.
Cell-free Infection Experiments
Purified resting CD4+ T cells were spinoculated with concentrated HIVNL4-3 or
infected without spinoculation. The cells were then washed and resuspended
in medium containing SQV to prevent HIV spread. Control wells were treated
with EFV. Cells were incubated for 3 days and stimulated with anti-CD3/
CD28 beads (Invitrogen), IL-2, and IL-7 and in the presence of raltegravir for
24 hr prior to flow cytometry analysis of HIV-1 Gag expression. Parallel cultures
were used for monitoring HIV integration by Alu-PCR.
Flow Cytometry
Prior to infection by co-culture, target resting CD4+ T cells were stained with
eFluor670 (eBioscience). To monitor HIV-1 Gag expression, co-cultures
were fixed with BD CytoFix/CytoPerm buffer (BD Biosciences) and washed
with Perm/Wash buffer (BD Biosciences). Cells were stained with anti-HIV-1
Gag antibody in BD Perm/Wash. The cells were then washed with BD Perm/
Wash buffer, resuspended in fluorescence-activated cell sorting (FACS)
buffer, and analyzed by flow cytometry. When monitoring for T cell activation,
the cells were stained for CD25, CD69, and HLA-DR expression. Viability was
monitored by staining cells with the Zombie Fixable Viability Kit (BioLegend).
Microscopy
Activated CD4+ T cells were spinoculated with HIVNL4-3-Gag-GFP (Jin et al.,
2009), washed, and incubated for 24 hr at 37 C. Typically, we obtained
about 10%–20% GFP-positive cells with our virus stock. Before
co-culturing with target cells, HIV-infected cells were washed. Infected
cells were mixed with resting CD4+ T cells in medium at a 2:1 ratio. Resting
CD4+ T cells were stained with CMAC (Molecular Probes) prior to
co-culture. The mixed cells were plated in poly-L-lysine-coated imaging
dishes and incubated at 37 C for 2 hr. Following incubation, the
co-cultured cells were washed and stained for CD4. Finally, the cells
were washed, fixed with paraformaldehyde, and imaged by confocal
microscopy.
Statistical Analysis
All experiments were repeated at least three times. All data are presented as
mean values ± SD. p values were calculated based on the Mann-Whitney
U test or Student’s t test (large sample size) using SPSS software. * denotes
p < 0.05, ** denotes p < 0.01, and NS denotes non-significant.
SUPPLEMENTAL INFORMATION
Supplemental Information includes Supplemental Experimental Procedures,
one figure, and three videos and can be found with this article online at
https://doi.org/10.1016/j.celrep.2018.07.079.
ACKNOWLEDGMENTS
This work was funded by the amfAR Mathilde Krim Fellowship in Basic
Biomedical Research (award 109263-59-RKRL) and the Immunology Training
Grant T32-AI007309. Additional funding was provided by the NIH (awards
AI084096, AI097117, and AI118682), the Inflammation Training Grant T32-
AI89673-5, and the Providence-Boston Center for AIDS Research (CFAR)
(P30-AI042853). We thank Chi Chan and David Levy for advice on transfec-
tions. We thank Nathan Roy, Caitlin Miller, Hisashi Akiyama, and Suryaram
Gummuluru for advice on imaging cell-cell contacts and reagents. We thank
Brian Tilton and the Boston University School of Medicine Flow Cytometry
Core for support with cell sorting and flow cytometry analysis. We thank
Michael Kirber and the Boston University School of Medicine Cellular Imaging
Core for microscopy support.
AUTHOR CONTRIBUTIONS
Conceptualization, L.M.A. and A.J.H.; Investigation, L.M.A. and M.B.H.;
Writing – Original Draft, L.M.A. and A.J.H.; Supervision, W.M. and A.J.H.;
Funding Acquisition, L.M.A., W.M., and A.J.H.
DECLARATION OF INTERESTS
The authors declare no competing interest.
Received: January 25, 2018
Revised: June 1, 2018
Accepted: July 22, 2018
Published: August 21, 2018
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