Article

An active HIV reservoir during ART is associated with

maintenance of HIV-specific CD8+ T cell magnitude
and short-lived differentiation status
Graphical abstract Authors
Hiroshi Takata, Julie L. Mitchell,
Julian Pacheco, ..., Lydie Trautmann,
RV254/SEARCH010, RV304/
SEARCH013

Correspondence
ltrautmann@global-id.org

In brief
Takata et al. found that the residual
antigen expression by the active HIV
reservoir during suppressive
antiretroviral therapy correlated with
increased HIV-specific CD8+ T cell
magnitude but prevented differentiation
into functional cells. Thus, targeting the
residual HIV reservoir may improve HIV-
specific CD8+ T cell functionality needed
for an HIV remission.

Highlights
d Magnitude of CD8+ T cell response associated with active HIV
reservoir size on therapy

d Sustained CD8+ T cell responses correlated with a greater HIV

provirus decay

d Residual HIV reservoir levels associated with maintenance of

short-lived CD8+ T cells

d Low HIV reservoir associated with generation of functional

HIV-specific CD8+ T cells

Takata et al., 2023, Cell Host & Microbe 31, 1494–1506

September 13, 2023 ª 2023 The Author(s). Published by Elsevier Inc.
https://doi.org/10.1016/j.chom.2023.08.012 ll
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Article
An active HIV reservoir during ART is associated
with maintenance of HIV-specific CD8+ T cell
magnitude and short-lived differentiation status
Hiroshi Takata,1,2,3 Julie L. Mitchell,1,2,3 Julian Pacheco,1 Amélie Pagliuzza,4 Suteeraporn Pinyakorn,2,3
Supranee Buranapraditkun,5 Carlo Sacdalan,6,5 Louise Leyre,4 Sam Nathanson,1 Juyeon C. Kakazu,2,3 Jintana Intasan,6
Peeriya Prueksakaew,6 Nitiya Chomchey,6 Nittaya Phanuphak,7 Mark de Souza,6 Elias K. Haddad,8 Morgane Rolland,2,3
Sodsai Tovanabutra,2,3 Sandhya Vasan,2,3 Denise C. Hsu,2,3 Nicolas Chomont,4 Lydie Trautmann,1,2,3,9,*
and RV254/SEARCH010, RV304/SEARCH013
1Vaccine and Gene Therapy Institute, Oregon Health & Science University, Beaverton, OR 97006, USA
2U.S. Military HIV Research Program, Walter Reed Army Institute of Research, Silver Spring, MD 20910, USA
3Henry M. Jackson Foundation for the Advancement of Military Medicine, Inc., Bethesda, MD 20817, USA
4Centre de Recherche du CHUM and Department of Microbiology, Infectiology and Immunology, Université de Montréal, Montréal, QC,

Canada
5Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand
6SEARCH Research Foundation, Bangkok, Thailand
7Institute of HIV Research and Innovation, Bangkok, Thailand
8Department of Medicine, Division of Infectious Diseases and HIV Medicine, Drexel University, Philadelphia, PA 19102, USA
9Lead contact

*Correspondence: ltrautmann@global-id.org
https://doi.org/10.1016/j.chom.2023.08.012

SUMMARY

Before initiation of antiretroviral therapy (ART), HIV-specific CD8+ T cells are dysfunctional and short lived. To
better understand the relationship between the HIV reservoir in CD4+ T cells and the magnitude and differen-
tiation status of HIV-specific CD8+ T cells, we investigated these cells from acute and chronic HIV-infected
individuals after 2 years of ART. Although both the HIV reservoir and the CD8+ T cell responses declined
significantly after 2 years of ART, sustained HIV-specific CD8+ T cell responses correlated with a greater
reduction of integrated HIV provirus. However, the magnitude of CD8+ T cells specific for HIV Gag, Pol,
Nef, and Vif proteins positively associated with the active reservoir size during ART, measured as cell-asso-
ciated RNA. Importantly, high HIV DNA levels strongly associate with maintenance of short-lived HIV-specific
CD8+ T cells, regardless of ART initiation time. Our data suggest that the active reservoir maintains HIV-spe-
cific CD8+ T cell magnitude but prevents their differentiation into functional cells.

INTRODUCTION ing of the HIV reservoir.11 However, HIV-specific CD8+ T cells

Although antiretroviral therapy (ART) can lead to viral suppression
below the limit of detection of clinical assays, life-long ART is
associated with significant burdens and comorbidities.1 ART-
free human immunodeficiency virus (HIV) remission, while desir-
able, has only been achieved in a few individuals, including five
bone-marrow transplant recipients and at least two natural con-
trollers with extremely low HIV reservoir.2 Rare people with HIV
(PWH), termed ‘‘elite controllers,’’ can naturally control HIV repli-
cation below the limit of routine clinical assays in the absence of
ART.3,4 HIV-specific CD8+ T cells with high functionality have
been considered key to achieving HIV control in elite control-
lers.5–8 During acute HIV infection (AHI), HIV-specific CD8+
T cells have been shown to limit viral replication,9–11 and we
have also previously shown that when ART was initiated in AHI
HIV-specific CD8+ T cells are functional and able to reduce seed-
become dysfunctional in most PWH during untreated chronic
HIV infection (CHI) because of continuous HIV antigen stimula-
tion, which induces the overexpression of checkpoint proteins
transducing inhibitory signals and drives cell exhaustion.12–15
ART initiated during CHI partially restores the defect in HIV-
specific CD8+ T cells by decreasing activation and expression
of checkpoint proteins, increasing cytokine production, and
increasing expression of the interleukin (IL)-7 receptor (IL-7R),
which reflects long-lived potential.16–21 However, the cells are
still dysfunctional compared with those in elite controllers, sug-
gesting that ART is not enough to fully reverse the defects.5–7,22
We recently showed that initiation of ART in AHI, but not CHI,
promoted the differentiation of HIV-specific CD8+ T cells with
self-renewal and long-lived phenotypes.23 Although ART low-
ered the checkpoint protein programmed cell death 1 (PD-1)
expression on ex vivo HIV-specific CD8+ T cells in people who

1494 Cell Host & Microbe 31, 1494–1506, September 13, 2023 ª 2023 The Author(s). Published by Elsevier Inc.
This is an open access article under the CC BY-NC license (http://creativecommons.org/licenses/by-nc/4.0/).

Article
Table 1. Clinical characteristics of study participants analyzed on ART
Chronic treated for
Pre-ART HIV infection stage 24 weeks (n = 17)
Percentage of male 100%
Age (years), median (interquartile range [IQR]) 29 (26–32)
Pre-ART plasma viral load copies/mL, median (IQR) 28,962 (7,127–92,155)
CD4 count cells/mm3 in plasma, median (IQR) 793 (603–949)
CD8 count cells/mm3 in plasma, median (IQR) 1,283 (780–1,658)
initiated ART during CHI, it remained higher relative to those in
people treated from AHI, and the overexpression of PD-1 was
further enhanced after in vitro antigen recall. Consistent with
this, recalled HIV-specific CD8+ T cells from people treated
from CHI showed dampened cell expansion and cytotoxic ca-
pacities compared with those from people treated from AHI, indi-
cating that HIV-specific CD8+ T cells retain functional defects
upon recall.23 Persistence of this residual dysfunction on ART
could be due to its imprinting in HIV-specific CD8+ T cells prior
to ART initiation or due to ongoing mechanisms that maintain
this dysfunction despite suppressive ART, though these have
not been demonstrated yet in HIV-specific CD8+ T cells.
Although the majority of HIV proviruses are defective in peo-
ple on ART,24 these defective proviruses still produce HIV anti-
gens, which are recognized by HIV-specific CD8+ T cells.25,26
Indeed, HIV Nef-specific CD8+ T cell responses positively asso-
ciated with the HIV reservoir size on long-term ART.27 The
change in magnitude of this Nef-specific response also corre-
lated with the HIV reservoir size on ART.28 These data suggest
that HIV-specific CD8+ T cells recognize HIV antigens but do
not lead to the elimination of the HIV reservoir under ART.
This is in line with the hypothesis that reactivated latently in-
fected cells are resistant to killing by HIV-specific CD8+
T cells29 and those with intact proviruses are enriched in cells
expressing immune checkpoint markers.30 However, the HIV
reservoir is defined as a compartment with a diverse virologic
profile, ranging from latency through viral protein expression
to virion production. It is unclear whether HIV-specific CD8+
T cells can recognize these different forms of HIV-infected cells
and eliminate them.
ART initiation during AHI leads to a lower HIV reservoir size31,32
and induces functionally superior HIV-specific CD8+ T cells
compared with those generated when ART is initiated during
CHI.23 As HIV-specific CD8+ T cells are short-lived effectors
with a transitional memory phenotype prior to ART initiation in
both AHI and CHI, we hypothesized that it is the HIV reservoir
that persists during ART that contributes to the residual dysfunc-
tion of HIV-specific CD8+ T cells and prevents their differentiation
into long-lived cells under ART. To better understand the
relationship between the HIV reservoir in CD4+ T cells and the
magnitude and differentiation status of HIV-specific CD8+
T cells, we investigated these cells after 2 years of ART in people
who initiated ART during AHI and CHI from the RV254/
SEARCH010 AHI cohort11,33,34 and the RV304/SEARCH013
CHI cohort,35 respectively, in Bangkok, Thailand. We measured
total and integrated HIV DNA and cell-associated RNA levels in
CD4+ T cells as well as the long-lived and stemness phenotypes
of HIV-specific CD8+ T cells within the same participants to define
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Chronic treated for Acute treated for
96 weeks (n = 17) 96 weeks (n = 48)
100% 98%
30 (28–34) 27 (24–31)
28,962 (7,127–92,155) 1,001,493 (64,465–7,349,735)
680 (548–858) 618 (489–806)
974 (599–1,195) 644 (462–785)
whether large or active HIV reservoirs prevent the differentiation
of functional HIV-specific CD8+ T cells under suppressive ART.
RESULTS
Frequencies of HIV-specific CD8+ T cells and HIV
reservoir size declined during ART
To understand the dynamics of the HIV reservoir and HIV-specific
CD8+ T cell response after ART initiation, we analyzed longitudi-
nal samples from participants in the RV304/SEARCH013 cohort
after 24 and 96 weeks of ART initiated during CHI (Table 1). All
participants were virologically suppressed with plasma viral
load below 50 copies/mL at these visits as well as at weeks 48
and 72. Both total HIV DNA and integrated HIV DNA copy
numbers in ex vivo CD4+ T cells significantly declined between
24 and 96 weeks of ART (Figure 1A). We analyzed frequencies
of HIV-specific CD8+ T cells recognizing all HIV proteins by stim-
ulating them with peptide pools tailored to the Thai cohort
(Table S1) and performing a new, more sensitive activation
induced marker (AIM)/intracellular cytokine staining (ICS) assay,
which consists of simultaneous ICS for interferon (IFN)-g and
detection of the AIMs CD69 and 4-1BB (Figure S1).36,37 HIV-spe-
cific CD8+ T cells from PWH on ART for 24 weeks targeted pre-
dominantly Env, Gag, Pol, and Nef, and the hierarchy of targeted
HIV proteins was maintained after 96 weeks of ART (Figures 1B
and S2A). When normalized for the number of peptides included
for each protein, responses were dominant for Gag and Nef
(Figures S2B and S2C). Frequencies of Gag-, Nef-, Rev/Tat-,
and total HIV-specific CD8+ T cells significantly declined between
24 and 96 weeks of ART, with a similar trend toward reduction in
the response to Env and Vif proteins (Figure 1C). HIV-specific
CD4+ T cells detected as CD69+CD40L+IFN-g+ cells from PWH
on ART prominently targeted Gag and Nef (Figures S1 and
S3A–S3D), and their responses did not exhibit a clear decline be-
tween 24 and 96 weeks of ART, suggesting that HIV-specific
CD4+ T cells are maintained differently on ART compared with
HIV-specific CD8+ T cells (Figure S3E). Taken together, these re-
sults indicate that the size of the HIV reservoir and the frequency
of HIV-specific CD8+ T cells sharply decline during the first 2
years of suppressive therapy initiated during CHI.
Sustained HIV-specific CD8+ T cell responses
associated with a reduction in the proviral HIV reservoir
To determine whether HIV-specific CD8+ T cells persisting dur-
ing ART contribute to the reduction in cells harboring HIV, we
analyzed the association between the frequency of HIV-specific
CD8+ T cells at 96 weeks of ART and the fold reduction in HIV
reservoir size (total HIV DNA and integrated HIV DNA) between
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A B

Figure 1. HIV reservoir size and frequency of HIV-specific CD8+ T cell decay after ART initiation in people treated from CHI
(A) Total and integrated HIV DNA copy number in CD4+ T cells from paired samples 24 and 96 weeks after ART initiation in people treated from CHI.
(B) Frequency of CD69+4-1BB+IFN-g+ HIV-specific CD8+ T cells in total CD8+ T cells 96 weeks after ART initiation.
(C) Frequency of CD69+4-1BB+IFN-g+ HIV-specific CD8+ T cells in total CD8+ T cells 24 and 96 weeks after ART initiation. Differences between 24 and 96 weeks
after ART initiation were analyzed by Wilcoxon test. **p < 0.01; ***p < 0.001.

24 and 96 weeks of ART. Strikingly, higher reduction of inte-
grated HIV DNA levels correlated with a higher frequency of
Nef-specific CD8+ T cells and a trend toward higher frequencies
of Gag- and total HIV-specific CD8+ T cells (Figures 2A and 2B).
Moreover, the fold reduction in the HIV reservoir size also corre-
lated with a smaller decline in the frequency of Gag-, Nef-, Vif-,
Rev/Tat-, and total HIV-specific CD8+ T cells (Figures 2C and
2D), calculated as fold change in the frequency of HIV-specific
CD8+ T cells between 24 and 96 weeks of ART. Similarly, the
fold reduction in the HIV reservoir size also correlated with fold
change in the frequencies of Vif- and total HIV-specific CD4+
T cells (Figure 2E). These results suggest that persistent HIV-
specific CD8+ and CD4+ T cells contribute to the elimination of
HIV-infected cells during suppressive ART. Of note, most of
these associations were lost when total HIV DNA was used as
a marker of the HIV reservoir, which may reflect the fact that
this assay captures long terminal repeat (LTR) circles and uninte-
grated HIV DNA38,39 that cannot generate viral proteins (Fig-
ure S4). These data suggest that only cells harboring integrated,
and potentially translation competent genomes are sensitive to
the pressure exerted by HIV-specific CD8+ T cells.
People treated in AHI have lower HIV reservoir but
comparable frequencies of HIV-specific CD8+ T cells
responding to antigen stimulation compared with
individuals treated in chronic infection
To better assess the virologic profile of HIV-infected cells that
can be recognized by HIV-specific CD8+ T cells, we measured
cell-associated HIV RNA (LTR-gag transcripts) as a surrogate
of the active reservoir. As most people who initiated ART during
CHI typically display a high but narrow range in the frequency of
infected cells (100–1,000 copies of HIV DNA per million CD4+

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A Figure 2. Higher reduction of the HIV reservoir associated

with higher and stable frequencies of HIV-specific CD8+
and CD4+ T cells on suppressive ART
(A and B) Correlation between the fold change in integrated HIV DNA
copy number and the frequency of CD69+4-1BB+IFN-g+ cells
directed against Nef and Gag (A) as well as total HIV-specific CD8+
T cells (B) in total CD8+ T cells from 96 weeks after ART initiation.
(C and D) Correlation between the fold change in integrated HIV
DNA copy number and the fold change in frequency of CD69+4-
1BB+IFN-g+ total HIV-specific CD8+ T cells in total CD8+ T cells
(C) and HIV-specific CD8+ T cells directed against Gag, Nef, Vif, and
Rev/Tat (D) from 24 to 96 weeks after ART initiation.
(E) Correlation between the fold change in integrated HIV DNA copy
B C number and the fold change in frequency of CD69+CD40L+IFN-g+
total HIV-specific CD4+ T cells in total CD4+ T cells and HIV-specific
CD4+ T cells directed against Vif from 24 to 96 weeks after ART
initiation. Correlations were analyzed by Spearman correlation with
the Benjamini-Hochberg procedure for multiple comparisons (false
discovery rate [FDR] < 0.1). Underlined p value represents
FDR > 0.1. *p < 0.05; **p < 0.01; ****p < 0.0001.

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A B

Figure 3. HIV reservoir size and frequency of HIV-specific CD8+ T cells under suppressive ART in people treated from AHI and CHI
(A) Total HIV DNA and cell-associated RNA copy number in CD4+ T cells 96 weeks after ART initiation in people treated from AHI (Tx AHI) and CHI (Tx CHI). Open
circles represent values under the detection limit.
(B) Correlation between the total HIV DNA and cell-associated RNA copy numbers in CD4+ T cells 96 weeks after ART initiation in people treated from AHI
and CHI.

(legend continued on next page)

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T cells), we analyzed samples from people who initiated ART
during AHI to provide a wider range of HIV reservoir size. As pre-
viously reported, people who initiated ART during AHI showed
significantly lower levels of total HIV DNA31,32 and cell-associ-
ated HIV RNA levels compared with people who initiated ART
during CHI (Figure 3A). Of note, these two HIV reservoir mea-
sures strongly correlated (Figure 3B). Unfortunately, measure-
ment of integrated HIV DNA levels could not be done concom-
itant with measurement of the active reservoir, as the nucleic
acid extraction process causes shearing of the DNA into
fragments too small for the long-range Alu PCR used for HIV
integrated DNA. Although more than half of the participants
who initiated ART during AHI had no detectable levels of
cell-associated HIV RNA (Figure 3A), HIV-specific CD8+
T cells were detected in all participants with a dominance for
Env and Pol HIV proteins, but no protein dominance when ad-
justing for the number of peptides per protein (Figure 3C).
Though people treated from AHI had lower HIV reservoir levels,
the frequency of HIV-specific CD8+ T cells responding to anti-
gen stimulation was comparable to those from people treated
from CHI (Figure 3D).
HIV transcription associated with the frequency of HIV-
specific CD8+ T cells during ART
To analyze whether the HIV reservoir burden affects HIV-specific
CD8+ T cell frequencies and further understand the extent
to which the HIV reservoir size affects HIV-specific CD8+ T cell
responses during ART, we performed correlative analyses be-
tween the different measures of HIV reservoir and the magnitude
of total HIV- or HIV protein-specific CD8+ T cell responses. Total
HIV DNA levels positively correlated with Gag-, Pol-, and total
HIV-specific CD8+ T cells with R values similar to those previ-
ously reported for Nef responses (Figures 4A and S5A).27 The
correlations were further adjusted for the groups (Tx AHI and
Tx CHI) and pre-ART plasma viral load because those potentially
bias the HIV-specific CD8+ T cell responses and the HIV reservoir
size, respectively. Pol-specific CD8+ T cells were still weakly
associated with total HIV DNA copies after the adjustment.
Moreover, cell-associated HIV RNA levels correlated with the
frequencies of HIV-specific CD8+ T cells recognizing all HIV pro-
teins except for Env as well as total HIV-specific CD8+ T cell fre-
quencies, with stronger associations compared with those for
total HIV DNA, and the correlations with Gag-, Pol-, Nef-, Vif-,
and total HIV-specific CD8+ T cells remained significant after
adjustment for group and pre-ART viral load (Figures 4B and
S5B). Consistently, transcriptional activity of the HIV reservoir,
calculated as the ratio of cell-associated HIV RNA to HIV DNA
copy number, showed similar associations with frequencies of
HIV-specific CD8+ T cells as the cell-associated RNA levels
(Figures 4C and S5C). These results demonstrate that the magni-
tude of HIV-specific CD8+ T cells persisting on ART is associated
with the active HIV reservoir size, suggesting that they may be
maintained by residual viral transcription from the active HIV
reservoir during ART.
(C) Frequency of CD69+4-1BB+IFN-g+ HIV-specific CD8+ T cells in total CD8+ T cells from people treated from acute HIV infection 96 weeks after ART initiation,
with and without normalization per number of peptides corresponding to each HIV protein.
(D) Comparison of the frequency of CD69+4-1BB+IFN-g+ HIV-specific CD8+ T cells in people treated from CHI and AHI. Differences between people treated from
AHI and CHI were analyzed by Mann-Whitney test. Correlation was analyzed by Spearman correlation. *p < 0.05; **p < 0.01; ****p < 0.0001.
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HIV reservoir size associated with the differentiation
status of HIV-specific CD8+ T cells persisting on ART
To characterize the differentiation status of HIV-specific CD8+
T cells persisting on ART, we used peptide major histocompati-
bility complex (MHC) class I tetramers specific for 5 different
HIV epitopes restricted by human leukocyte antigen (HLA)-
A*1101 and Cw*0102, dominant alleles in the Thai popula-
tion.11,23,40 Tetramer+ HIV-specific CD8+ T cells after 96 weeks
of ART did not associate with pre-ART viral load (Figure 5A) or
pre-ART total HIV DNA (Figure S6A). However, the frequency
of HIV-specific CD8+ T cells detected by tetramer significantly
correlated with total HIV DNA on ART, while the association
and strong trend were also confirmed within people treated
from AHI and CHI, respectively (Figure 5B). Two transcription
factors, T cell factor 1 (TCF-1) and thymocyte selection-associ-
ated high-mobility group box (TOX), have been shown to play
a role in self-renewal capacity41–43 and cell exhaustion,44–48
respectively, during chronic viral infections, and their expression
is used to classify CD8+ T cells as self-renewing cells (TCF-1high
TOXlow) and short-lived cells (TCF-1lowTOXhigh).49 Prior to ART
initiation, HIV-specific CD8+ T cells were almost exclusively
TCF-1lowTOXhigh short-lived effector cells, regardless of the
stage of infection (Figures S6B and S6C). After ART initiation,
the majority of HIV-specific CD8+ T cells still remained TCF-1low
TOXhigh short-lived effector cells in people who initiated ART
during CHI, whereas the cells from people treated from AHI
differentiated into TCF-1highTOXlow stem-like self-renewing cells
(Figures 5C and 5D). Similar to the frequency of HIV-specific
CD8+ T cells, the frequency of TCF-1highTOXlow cells did not
correlate with pre-ART HIV DNA (Figure S6D). In contrast, total
HIV DNA levels on ART negatively correlated with the fre-
quencies of TCF-1highTOXlow HIV-specific CD8+ T cells, while
the same correlation and trend were also found in people treated
from CHI and AHI, respectively (Figure 5E). These data suggest
that the HIV reservoir size affects the balance of these popula-
tions, with a higher HIV reservoir size associated with mainte-
nance of a short-lived effector phenotype and a lower HIV reser-
voir size associated with more differentiation toward stem-like
self-renewing cells. We further investigated the cell exhaustion/
activation status, measured by PD-1 expression, and long-lived
capacity, measured by IL-7R expression, of HIV-specific CD8+
T cells during ART. Expression of these markers after ART did
not associate with pre-ART HIV DNA levels (Figure S6E).
However, higher HIV DNA levels on ART associated with higher
PD-1 expression on HIV-specific CD8+ T cells, and the associa-
tion and strong trend were also found in people treated from
CHI and AHI, respectively (Figure 5F), although this correlation
was not significant after adjustment. Conversely, lower HIV
DNA levels associated with higher expression of IL-7R on
HIV-specific CD8+ T cells, and the association and strong trend
were also confirmed in people treated from CHI and AHI,
respectively. Most of these correlations were maintained when
cell-associated HIV RNA was used instead of total HIV DNA,
although they lost significance when they were adjusted
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A Figure 4. Frequency of HIV-specific CD8+
C
for timing of ART initiation and pre-ART plasma viral load
(Figures S7A–S7C). Altogether, these findings suggest that a
larger HIV reservoir prevents the differentiation of HIV-specific
CD8+ T cells into long-lived HIV-specific CD8+ T cells with self-
renewal capacity on ART.
DISCUSSION
In this study, we measured total and integrated HIV DNA as well as
cell-associated HIV RNA through 96 weeks of ART to identify asso-
ciations with HIV-specific CD8+ T cell responses in people treated
in AHI and CHI. We found that although both the HIV reservoir
levels and HIV-specific CD8+ T cell responses decline between
weeks 24 and 96 in people treated in CHI, sustained HIV-specific
CD8+ T cell responses on ART correlated with a reduction in inte-
1500 Cell Host & Microbe 31, 1494–1506, September 13, 2023
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T cells associated with size of active HIV
reservoir on ART
Correlation between the frequency of CD69+4-
1BB+IFN-g+ HIV-specific CD8+ T cells in total
CD8+ T cells and the total HIV DNA (A), cell-
associated RNA (B), or ratio of cell-associated
RNA to HIV DNA (C) copy number in CD4+ T cells
96 weeks after ART initiation. Open circles repre-
sent values under the detection limit. Correlations
were analyzed by Spearman correlation with the
Benjamini-Hochberg procedure for multiple com-
parisons (FDR < 0.1). Underlined p value repre-
sents FDR > 0.1. p values adjusted for group (Tx
AHI and Tx CHI) and pre-ART plasma viral load are
also shown. *p < 0.05; **p < 0.01.
grated HIV DNA. This is consistent with our
previous report that HIV-specific CD8+
T cells persisting shortly after ART initiation
in AHI more efficiently restrict the seeding
of the HIV reservoir.11 These data suggest
that strategies aimed at maintaining HIV-
specific CD8+ T cells after ART initiation,
either early or later in infection, may lead
to a greater reduction of the pool of in-
fected cells.50 Importantly, we found sig-
nificant correlations between the size of
the transcriptionally active reservoir,
measured by cell-associated RNA, and
the magnitude of HIV-specific CD8+ T cell
responses directed against total HIV pro-
teins as well as individual Gag, Pol, Nef,
and Vif proteins. These data suggest that
transcriptionally active HIV-infected cells
may still produce antigens from all HIV
proteins, possibly in viral sanctuary sites
such as the genital tract and lymphoid
tissues.51,52
In chronic infections and cancer, CD8+
T cells with stem-like self-renewal capac-
ity play a critical role in disease outcome.
These cells express the transcriptional
factor TCF-1 and can give rise to
more differentiated TCF-1low short-lived cells.43 Indeed, these
TCF-1+ cells are the predominant cell type that proliferates in
response to anti-PD-1 immune checkpoint blockade ther-
apy53,54 and are associated with longer survival in cancer pa-
tients.55 Moreover, HIV-specific CD8+ T cells in elite controllers
have higher proliferative capacity5 and elevated TCF-1 expres-
sion compared with non-controllers.22,47 We observed that a
larger HIV reservoir size is associated with reduced differentia-
tion of TCF-1highTOXlow stem-like self-renewing cells and main-
tenance of TCF-1lowTOXhigh short-lived effector cells. Similarly,
we found that higher total HIV DNA levels were associated with
lower expression of IL-7R and maintenance of PD-1 expression
on HIV-specific CD8+ T cells. Although we detected very strong
correlations between the differentiation status of HIV-specific
CD8+ T cells on ART and the HIV reservoir size on ART, we did
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A B Figure 5. The HIV reservoir size associated with differ-

entiation status of HIV-specific CD8+ T cells on ART
(A and B) Correlation between frequency of ex vivo pMHC
tetramer+ HIV-specific CD8+ T cells in total CD8+ T cells from
people treated for more than 1.5 years from AHI and CHI and
Pre-ART plasma viral load (A) or total HIV DNA copies per 106
CD4+ T cells (B) at the sampling of HIV-specific CD8+ T cells
analyzed.
(C and D) Frequency of TCF-1highTOXlow and TCF-1lowTOXhigh
cells in HIV-specific CD8+ T cells from people treated from AHI
and CHI.
(E) Correlation between total HIV DNA copies and frequency of
TCF-1highTOXlow cells in HIV-specific CD8+ T cells on ART.
(F) Correlation between total HIV DNA copies and expression
of PD-1 and IL-7R on ex vivo HIV-specific CD8+ T cells on ART.
Correlations were analyzed by Spearman correlation with the
Benjamini-Hochberg procedure for multiple comparisons
C (FDR < 0.1). Differences between people treated from AHI and
CHI were analyzed by Wilcoxon test. p values adjusted for
group (Tx AHI and Tx CHI) and pre-ART plasma viral load are
also shown. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

D E

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not observe these associations with HIV DNA levels prior to ART
initiation. This suggests that it was not an imprinting of the anti-
gen burden before ART initiation but rather an active recognition
of HIV antigens by CD8+ T cells during ART that was responsible
for these associations, although PD-1 expression could poten-
tially be regulated by epigenetic programming.19 Together, these
data suggest that higher HIV reservoir size on ART prevents the
differentiation of functional stem-like self-renewing HIV-specific
CD8+ T cells, which in turn may prevent a functional recall
response by HIV-specific CD8+ T cells after ART interruption
by sustaining a skewed differentiation toward short-lived cells.
Previous studies which measured HIV-specific CD8+ T cell fre-
quencies during ART showed that they predominantly target
Gag, Pol, and Nef proteins27,56,57 and have reported an associa-
tion with the HIV reservoir size only for Nef responses.27,28 In our
study, Nef- and Pol-specific CD8+ T cell responses were more
strongly associated with cell-associated RNA level than Gag-
specific CD8+ T cell responses, suggesting that Gag-specific
CD8+ T cells are less efficient at sensing ongoing HIV activation
than Nef- or Pol-specific CD8+ T cells. If this finding is confirmed
by additional studies, it may have important implications on
therapeutic vaccines that may need to target CD8+ T cells
specific for Nef and Pol in addition to Gag to contain residual
HIV activation under ART. We also found a dominance of Env re-
sponses in our cohorts and associations between cell-associ-
ated HIV RNA with several HIV proteins. As Env is more variable
across individuals than the other proteins,58 our use of a tailored
peptide pool covering the entire HIV consensus sequence in the
Thai cohorts as well as the major variants may have allowed for
detection of these Env responses. This difference of dominance
in responses could also be explained by the cohorts analyzed,
with our cohorts predominantly composed of Thai individuals
with different HLAs and infected with a clade CRF01_AE virus.
Finally, the assays used in previous studies and our study are
also different. The ELISpot, ICS, and AIM assays used in previ-
ous studies have a high limit of detection and may not be sensi-
tive enough to detect the low frequencies of HIV-specific CD8+
T cells that persist on ART.23 We designed a new assay that
combines AIM and ICS to lower the limit of detection and detect
these low frequencies of HIV-specific CD8+ T cells with higher
accuracy. This enhanced accuracy in measuring responses for
all HIV proteins may explain our ability to detect correlations be-
tween HIV reservoir and HIV-specific CD8+ T cell responses
directed to numerous HIV proteins. We used MHC class I tetra-
mers tailored to the Thai population to detect low frequencies of
HIV-specific CD8+ T cell responses. This method is the most
sensitive measurement of HIV-specific CD8+ T cells, detecting
all T cells specific for the tetramer regardless of their ability to
respond to peptide stimulation, and in turn provided the most
robust correlation between the magnitude of HIV-specific
CD8+ T cells and HIV reservoir. The use of different and novel as-
says to detect HIV-specific CD8+ T cells provided complemen-
tary data to draw a more complete picture of the association be-
tween the HIV reservoir persisting on ART and these responses.
We observed that the active HIV reservoir, measured as cell-
associated RNA, strongly associated with frequencies of HIV-
specific CD8+ T cells detected by the AIM/ICS assay but weakly
associated with frequencies of cells detected by the peptide
MHC (pMHC) tetramers. This discordance potentially came
1502 Cell Host & Microbe 31, 1494–1506, September 13, 2023
Article
from differences in the HIV-specific CD8+ T cells detected by
the 2 assays. The AIM/ICS assay detects HIV-specific CD8+
T cells that are able to respond to antigen stimulation by express-
ing the AIM markers and producing cytokines, whereas cells de-
tected by tetramers also contain HIV-specific CD8+ T cells that
are unable to respond due to possible cell exhaustion. These
observations suggest that the active HIV reservoir maintains
HIV-specific CD8+ T cells that are able to respond to HIV anti-
gens. Another possibility would be differences in specificity of
cells detected by the 2 assays, as tetramers only identify cells
recognizing a single epitope, whereas the AIM/ICS was de-
signed to identify any cells recognizing any epitopes in HIV.
Further, the cell-associated RNA assay detects LTR-gag RNA
and, while the AIM/ICS assay would detect HIV-specific CD8+
T cells targeting all HIV proteins, including Gag, only 20% of
HIV-specific CD8+ T cells detected by the tetramers were Gag-
specific, with the majority of cells being Nef-specific CD8+
T cells, which are immunodominant in this cohort.11,23,40 Future
studies are needed to evaluate direct associations between HIV
RNA expression and HIV-specific CD8+ T cells targeting the HIV
protein corresponding to the RNA measured.
In this study, we included people treated in AHI and CHI to
analyze a wider range of HIV reservoir size than studies analyzing
only people treated in CHI. However, even within people treated
in AHI, the HIV reservoir size varies greatly, from undetectable
HIV DNA levels to levels comparable to people treated in CHI.
As we previously showed that sustained HIV-specific CD8+
T cell responses shortly after ART initiation in AHI limited HIV
reservoir seeding,11 these differences in HIV reservoir size might
be due to differences in the potency of the immune response. We
chose to analyze HIV reservoir after 24 and 96 weeks of ART as
this corresponds to a time after the initial rapid decay of HIV
reservoir after ART initiation but before stabilization of the reser-
voir size after 4 years of ART.59,60 As we previously reported that
HIV-specific CD8+ T cell responses remain stable on long-term
ART over 3–5 years,61 this time frame also allowed us to measure
a decay in HIV-specific CD8+ T cell frequency prior to stabiliza-
tion of these responses. Previous studies, which only detected
an association between Nef-specific CD8+ T cell responses
and changes in the HIV reservoir size, were conducted using
samples collected after more than 4 years of ART, when the
decay of both HIV reservoir and HIV-specific CD8+ T cell re-
sponses is much smaller.27,28 In this study, the active reservoir
was measured by cell-associated HIV RNA. We acknowledge
that a large fraction of proviruses producing HIV transcripts are
likely to be defective. However, this form is more likely to pro-
duce HIV antigens that can be recognized by HIV-specific
CD8+ T cells,25 possibly explaining the stronger correlations
we measured between HIV-specific CD8+ T cells and cell-asso-
ciated RNA.
Our study suggests that residual immune dysfunction driven
by the active HIV reservoir on ART could contribute to the lack
of viral control after analytical treatment interruption by prevent-
ing the differentiation of functional stem-like self-renewing HIV-
specific CD8+ T cells that can mount efficient rapid recall re-
sponses. Therefore, HIV remission strategies will likely need to
target transcriptionally active proviruses producing viral proteins
during ART to harness HIV-specific CD8+ T cells to control re-
bounding of the virus after ART cessation. Future studies are

Article
needed to define strategies to reduce or silence the active HIV
reservoir on ART and define whether those would be able to
reverse the residual dysfunction of HIV-specific CD8+ T cells per-
sisting on ART.
Limitations of the study
This study has several limitations. It would have been informative
to determine correlations between HIV-specific CD8+ T cell re-
sponses and intact HIV DNA levels. Most participants in our
study were infected with HIV-1 CRF01_AE or CRF01_AE/B re-
combinants, and the intact proviral DNA assay (IPDA) has not
yet been adapted to CRF01_AE. In addition, the total numbers
of HIV genomes are very low in people treaded from AHI.31,61
Therefore, intact HIV genomes are present at extremely low fre-
quencies in the majority of these participants,62 suggesting the
IPDA assay might not be an optimal assay for this population.
However, both total and integrated HIV DNA levels have been
shown to strongly correlate with intact proviruses measured by
IPDA.63 Additionally, we believe that non-intact HIV-infected
cells expressing HIV protein,25,26 which would not be captured
by the IPDA assay, could also contribute to the altered differen-
tiation of functional HIV-specific CD8+ T cells on ART. A second
limitation of this study was the small sample size due to the lon-
gitudinal sampling and identified number of participants positive
for the pMHC tetramers used. It would be informative to analyze
correlations between HIV-specific CD8+ T cell responses and
HIV reservoir size in people treated from AHI and CHI separately;
however, the limited sample size in each participant group did
not confer enough statistical power to perform the analysis
with the separate groups. Therefore, we performed careful sta-
tistical analyses to eliminate possible biases by adjusting for
the groups and pre-ART plasma viral load. Nevertheless, we
were able to confirm that the majority of the correlations re-
mained significant after adjusting for participant group and
pre-ART plasma viral load using a multivariate linear regression
model. Additional studies are warranted to understand how
timing of ART initiation affects the association between HIV-spe-
cific CD8+ T cells and HIV reservoir persistence under ART.
STAR+METHODS
Detailed methods are provided in the online version of this paper
and include the following:
d KEY RESOURCES TABLE
d RESOURCE AVAILABILITY
B Lead contact
B Materials availability
B Data and code availability
d EXPERIMENTAL MODEL AND STUDY PARTICIPANT
DETAILS
B Study design
B Study approval
d METHOD DETAILS
B Antibodies and Reagents for Flow cytometry analysis
B HIV peptide pools for intracellular cytokine staining
+ +
B Ex vivo detection of HIV-specific CD8 and CD4
T cells by intracellular cytokine staining
B Flow cytometry analysis
ll
OPEN ACCESS
B HIV DNA and HIV RNA measures
B Statistical analysis
SUPPLEMENTAL INFORMATION
Supplemental information can be found online at https://doi.org/10.1016/j.
chom.2023.08.012.
ACKNOWLEDGMENTS
We thank our study participants and staff from the Thai Red Cross AIDS
Research Centre (TRC-ARC), Chulalongkorn University, and Armed Forces
Research Institute of Medical Sciences (AFRIMS) for their valuable contribu-
tions to this study. We want to thank D. Brooks and B. Colton for technical
assistance. We are grateful to the Government Pharmaceutical Organization
of Thailand, ViiV Healthcare, Gilead Sciences, and Merck for providing the anti-
retrovirals for this study. We also thank the RV254/SEARCH010 and RV304/
SEARCH013 study group members at TRC-ARC, AFRIMS, and MHRP. All
MHC class I peptide monomers were obtained through the NIH Tetramer
Core Facility. This study was supported by the following sources: NIH grants
R01AI108433, R01MH106466, and UM1AI64560, a cooperative agreement
(W81XWH-07-2-0067, W81XWH-11-2-0174, W81XWH-18-2-0040) between
the Henry M. Jackson Foundation for the Advancement of Military Medicine
Inc. and the U.S. Department of Defense, and, in part, by the Division of
AIDS, National Institute of Allergy and Infectious Diseases, National Institute
of Health (AAI20052001). The content of this manuscript is solely the responsi-
bility of the authors and does not necessarily represent the official views of any
of the institutions mentioned above, the U.S. Department of the Army or the
U.S. Department of Defense, the Henry M. Jackson Foundation for the
Advancement of Military Medicine, Inc., the National Institutes of Health,
the Department of Health and Human Services, or the United States Govern-
ment, nor does the mention of trade names, commercial products, or organiza-
tions imply endorsement by the Thai Red Cross AIDS Research Centre. The
investigators have adhered to the policies for protection of human participants
as prescribed in AR-70-25.
AUTHOR CONTRIBUTIONS
H.T. designed and performed the experiments, analyzed the data, and wrote
the manuscript. J.L.M., J.P., S.N., and J.C.K. performed the experiments and
helped analyze the data and write the manuscript. A.P. and L.L. performed
the HIV DNA and RNA quantifications. S.B., M.R., and S.T. performed the
sequence and alignment of HIV variants for designing the HIV peptide pools.
S.P. provided help in statistical analyses. C.S., J.I., S.T., P.P., and N. Chomchey
managed the participant recruitment and follow-up in the studies. N.P. and
M.d.S. provided support for the clinical studies. N. Chomont and E.K.H. pro-
vided conceptual advice and edited the manuscript. S.V. and D.C.H. designed
the clinical study, provided conceptual advice, and edited the manuscript. L.T.
designed the experiments, analyzed the data, and wrote the manuscript.
DECLARATION OF INTERESTS
The authors declare no competing interests.
Received: January 20, 2023
Revised: June 2, 2023
Accepted: August 14, 2023
Published: September 13, 2023
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STAR+METHODS

KEY RESOURCES TABLE

REAGENT or RESOURCE SOURCE IDENTIFIER

Antibodies
BV510 anti-human CD14 (clone M5E2) BioLegend Cat# 301842; RRID: AB_2561946
BV510 anti-human CD19 (clone SJ25C1) BioLegend Cat# 363020; RRID: AB_2564229
PE Cy5 anti-human CD127 (clone A019D5) BioLegend Cat# 351324; RRID: AB_10915554
Pacific Blue labelled anti-human IFN-g BioLegend Cat# 502522; RRID: AB_893525
(clone 4S.B3)
APC labelled anti-human 4-1BB BioLegend Cat# 309810; RRID: AB_830672
(clone 4B4-1)
Alexa Fluor 488 anti-human CD69 BioLegend Cat# 310916; RRID: AB_528869
(clone FN50)
APC Fire810 anti-human CD4 (clone SK3) BioLegend Cat# 344662; RRID: AB_2860884
Brilliant Violet 785 anti-human CD8 (clone BioLegend Cat# 301046; RRID: AB_2563264
RPA-T8)
PE labelled anti-human TCF-1 (clone BioLegend Cat# 655208; RRID: AB_2728492
7F11A10)
BV510 anti-human CD3 (clone UCHT 1) BD Biosciences Cat# 566105; RRID: AB_2739563
BUV496 anti-human CD8 (clone RPA-T8) BD Biosciences Cat# 612942; RRID: AB_2870223
BUV563 anti-human CD4 (clone SK3) BD Biosciences Cat# 612912; RRID: AB_2739451
BUV737 anti-human PD-1 (clone EH12.1) BD Biosciences Cat# 612791; RRID: AB_2870118
Alexa Fluor 488 anti-TOX Cell Signaling Technology Cat# 44682S
BD FastImmune Co-Stimulatory Antibodies BD Biosciences Cat# 347690; RRID: AB_647457
(CD28/CD49d)
Biological samples
Human peripheral blood mononuclear U.S. Military HIV Research Program RV254/ SEARCH010
cells (PBMCs)
Human peripheral blood mononuclear U.S. Military HIV Research Program RV304/ SEARCH013
cells (PBMCs)
Chemicals, peptides, and recombinant proteins
BD GolgiPlug Protein Transport Inhibitor BD Biosciences Cat# 555029
(Brefeldin A)
BD GolgiStop Protein Transport Inhibitor BD Biosciences Cat# 554724
(Monensin)
BD Perm/Wash Perm/Wash Buffer BD Biosciences Cat# 554723
BD Horizon Brilliant Stain Buffer BD Biosciences Cat# 566349
BD Horizon Brilliant Stain Buffer Plus BD Biosciences Cat# 566385
Foxp3 / Transcription Factor Staining Thermo Fisher Scientific Cat# 00-5523-00
Buffer Set
LIVE/DEAD Fixable Aqua Dead Cell Thermo Fisher Scientific Cat# L34957
Stain Kit
LIVE/DEAD Fixable Yellow Dead Cell Thermo Fisher Scientific Cat# L34959
Stain Kit
BV650 Streptavidin BioLegend Cat# 405232
HIV-1 CRF01_AE Fifteen-mer peptides Anaspec N/A, Custom
Critical commercial assays
EasySep Human CD4+ T Cell STEMCELL Technologies Cat# 19052
Enrichment Kit
QuantiNova Probe PCR Kit QIAGEN Cat# 80224
(Continued on next page)

Cell Host & Microbe 31, 1494–1506.e1–e4, September 13, 2023 e1

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Continued
REAGENT or RESOURCE SOURCE IDENTIFIER
SuperScript III One-Step RT-PCR System Invitrogen (ThermoFisher) Cat# 12574026
with Platinum Taq DNA Polymerase
AllPrep DNA/RNA/miRNA Universal KIT QIAGEN Cat# 80224
MEGAscript T7 Transcription Kit Ambion Cat# AMB13345
Oligonucleotides
ULF1 (Forward Pre-PCR): 5’- ATG CCA Vandergeeten et al.64 N/A
CGT AAG CGA AAC TCT GGG TCT CTC
TDG TTA GAC-3’
UR1 (Reverse Pre-PCR): 5’-CCA TCT CTC Vandergeeten et al.64 N/A
TCC TTC TAG C-3’
Lambda (l) T (Forward Post-PCR): 5’-ATG Vandergeeten et al.64 N/A
CCA CGT AAG CGA AAC T-3’
UR2 (Reverse Post-PCR): 5’-CTG AGG Vandergeeten et al.64 N/A
GAT CTC TAG TTA CC-3’
Software and algorithms
FlowJo v10.8.1 FlowJo, LLC N/A
Prism 9 GraphPad Software, LLC. N/A
Other
Northern Lights 3000 cytometer Cytek Biosciences NL-3000
In vitro synthetized RNA transcript This paper N/A
Rotor-Gene Q QIAGEN Cat# 9001620

RESOURCE AVAILABILITY

Lead contact
Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Lydie
Trautmann (ltrautmann@global-id.org).

Materials availability
This study did not generate new unique reagents.

Data and code availability

Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request. This
paper does not report original code.

EXPERIMENTAL MODEL AND STUDY PARTICIPANT DETAILS

Study design
Forty-eight people with HIV (PWH) from the RV254/ SEARCH010 study who initiated ART during AHI and 18 participants who
initiated ART during CHI from the RV304/ SEARCH013 study, all conducted in Bangkok, Thailand, were included in these
analyses (Table 1). In the RV254/ SEARCH010 study, samples from HIV test clients were tested by fourth- and third-generation
immunoassays and HIV nucleic acid test. The participants were classified into Fiebig I to V based on HIV nucleic acid test,
p24 antigen, and third-generation and Western blot immunoassays as previously described.33,65 For all of the experiments, lab
investigators were blinded to the participant ID and HIV infection stage at ART initiation (AHI stages or CHI) of specimens during
procedures so as not to bias measurements. The specimen information was recovered to perform statistical analyses. Outliers
were not excluded from the analyses. All participants were pre-screened for HLA-A1101/ Cw0102 restricted HIV-specific CD8+
T cells.

Study approval
All study participants provided written informed consent approved by the Chulalongkorn University and Walter Reed Army Institute of
Research institutional review boards.

e2 Cell Host & Microbe 31, 1494–1506.e1–e4, September 13, 2023

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METHOD DETAILS

Antibodies and Reagents for Flow cytometry analysis

BV510 labelled anti-CD14, BV510 labelled anti-CD19, PE Cy5 labelled anti-CD127, Pacific Blue labelled anti-IFN-g, APC labelled
anti-4-1BB, Alexa 488 labelled anti-CD69, APC Fire810 labelled anti-CD4, BV785 labelled anti-CD8, and PE labelled anti-TCF-1
came from Biolegend. BV480 labelled anti-CD3, BUV496 labelled anti-CD8, BUV563 labelled anti-CD4, BUV737 labelled anti-
PD-1, GolgiPlug, GolgiStop, Perm Wash Buffer, Brilliant Stain Buffer, and Brilliant Stain Buffer Plus came from BD Biosciences. Alexa
Fluor 488 labelled anti-TOX came from Cell Signaling Technology. LIVE/DEAD Fixable Dead Cell Stain Kits (Aqua and Yellow) and
FoxP3 / Transcription Factor Staining Buffer Set came from Thermo Fisher Scientific. HLA-A*1101 HIV-1 NEF (GAFDLSFFLK and
QVPLRPMTYK), HLA-A*1101 HIV-1 POL (AIFQSSMTKIL), HLA-Cw*0102 HIV-1 GAG (YSPVSILDI), and HLA-Cw*0102 HIV-1 ENV
(CTPAGYAIL) soluble biotinylated monomers were produced at NIH Tetramer Core Facility as previously described53 and tetramer-
ized with BV650-labeled Streptavidin (BioLegend).

HIV peptide pools for intracellular cytokine staining

A large collection of 1,057 overlapping peptides spanning the entire HIV proteome were designed based on available HIV sequence
alignments derived from the SEARCH10/RV254 and RV144 Thai cohorts and encompassing the consensus sequence and the most
frequent HIV variants. The detailed description of the 1,057 peptides is provided in Table S1. Fifteen-mer peptides, overlapping by 11
amino acids, were synthesized by Anaspec (Fremont, CA). Lyophilized peptides were reconstituted in dimethyl sulfoxide (DMSO) at
10mg/ml, aliquoted and stored at -80C. Single peptides were pooled in 13 pools corresponding to Nef (unique pool, n=91 peptides),
Gag (2 pools, n= 72 peptides each pool), Pol (3 pools, n=92 peptides each pool), Rev/Tat (unique pool, n=89 peptides), Vif (unique
pool, n=71 peptides), Vpu/Vpr (unique pool, n=70 peptides), Env (4 pools, 2 pools including n=80 peptides, 2 pools including n=78
peptides).

Ex vivo detection of HIV-specific CD8+ and CD4+ T cells by intracellular cytokine staining
Thawed peripheral blood mononuclear cells (PBMCs) from PWH were plated at 2x106 cells/well in 96 well deep well plate in RPMI
1640 media containing 10% FBS and Penicillin/Streptomycin (R10 media). The cells were stimulated with HIV peptide pools (1 mg of
each peptide/mL) in the presence of anti-CD28/49d (0.5 mg/mL) in R10 media and incubated for 1 hour at 37 C. The same amount of
DMSO as the HIV peptide pool was added for a negative control. GolgiPlug (1 mL/mL) and GolgiStop (0.5 mL/mL) were added to each
well and the cells were incubated at 37 C overnight (16 hrs).

Flow cytometry analysis

For analysis of cytokine producing T cells, peptide stimulated PBMCs were first stained with LIVE/DEAD Aqua for 10 minutes at room
temperature. Cells were stained for other cell surface markers in Brilliant Stain Buffer at 4 C for 20 minutes followed by 2 times wash
with PBS containing 2% FBS (washing buffer). Cells were fixed in PBS containing 2% formaldehyde (fix buffer) for 20 minutes at room
temperature. Cells were washed and resuspended in Perm/Wash buffer for 15 minutes at room temperature. Cells were stained for
intracellular cytokine markers in Perm/Wash buffer at room temperature for 30 minutes, followed by 2 washes with washing buffer
and then fixation in fix buffer. Fixed cells were analyzed using a Northern Lights 3000 cytometer (Cytek Biosciences). The frequency
of CD69+4-1BB+IFN-g+ HIV-specific CD8+ T cells in total CD8+ T cells and CD69+CD40L+IFN-g+ HIV-specific CD4+ T cells in total
CD4+ T cells was reported after subtracting the frequency of those cells in the culture with DMSO only.
For phenotypic analysis with peptide-MHC class I complex tetramer (pMHC tetramer), thawed PBMCs were first stained for pMHC
tetramer, cell surface markers, and then fixed/permeabilized for intracellular/intranuclear staining with FoxP3 / Transcription Factor
Staining Buffer Set as previously described.11,23
All flow cytometry data was analyzed with FlowJo v10.8.1 (FlowJo, LLC)

HIV DNA and HIV RNA measures

The frequencies of CD4+ T cells harboring integrated HIV DNA was measured by real time nested PCR using Alu primers together with
a primer annealing to the HIV LTR in purified CD4+ T cells obtained by negative magnetic selection (StemCell), as previously
described.64 Serial dilutions of ACH2 cells carrying a single copy of the HIV genome were used as a standard.
To measure total HIV DNA and HIV RNA, cellular DNA and RNA were co-extracted from isolated CD4+ T cells, using the AllPrep
DNA/RNA Mini Kit (Qiagen) according to the manufacturer’s instructions. The frequency of cells harbouring total HIV DNA (LTR-gag)
was quantified by real-time PCR on the extracted genomic DNA, as previously described.64 Serial dilutions of ACH2 cells were used
as a standard. Cell-associated RNA (LTR-gag) was quantified by real-time nested RT-PCR, using the same primers as in the DNA
assay. In a first PCR reaction, RNA were reverse-transcribed and pre-amplified using the Superscript III one-step RT-PCR System
(ThermoFisher) in a 25mL reaction containing 0.5mM of each primers, and 8.5mL of extracted RNA. The reverse transcription was per-
formed at 50 C for 30min, followed by a denaturation step at 94 C for 2min, and 16 cycles of: 15s at 94 C, 30s at 55 C, 1min at 68 C,
with a final elongation step of 5min at 68 C. For the nested PCR, the pre-amplified cDNA was amplified by real-time PCR on a Rotor-
Gene Q instrument, using the same protocol than for the HIV DNA PCR.64 In vitro transcribed RNA (generated from plasmids with

Cell Host & Microbe 31, 1494–1506.e1–e4, September 13, 2023 e3

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MEGAscript T7 Transcription Kit, ThermoFisher) were used as a standard for absolute quantification. LTR-gag transcripts were
expressed as copies per 106 cells using the CD3 copy numbers measured in the HIV DNA assay or as copies per viral genome using
the total HIV DNA quantification results.
The sample detection limit for the cell-associated RNA assay is provided as Table S2. All real time nested PCR reactions were per-
formed in triplicate. The means of triplicates were used for data analysis.

Statistical analysis
Statistical analyses were performed using the nonparametric Mann-Whitney test (2 groups) for group comparisons, and the Wilcoxon
matched-pairs signed-rank test to compare CD8+ T cell responses or HIV reservoir measurements from same participant between
2 visits. The nonparametric Spearman test was used for all of the correlation analyses, and the Benjamini-Hochberg procedure was
used to correct for multiple comparisons (FDR < 0.1). P-values were adjusted by group (Tx AHI and Tx CHI) and pre-ART viral load,
using a multivariate linear regression model.

e4 Cell Host & Microbe 31, 1494–1506.e1–e4, September 13, 2023