Skip to main content
eScholarship
Open Access Publications from the University of California
Refine Search

UCSF

UC San Francisco Previously Published Works bannerUCSF

Terminal differentiation of T cells is strongly associated with CMV infection and increased in HIV-positive individuals on ART and lifestyle matched controls

Published Web Location

https://doi.org/10.1371/journal.pone.0183357Creative Commons 'BY' version 4.0 license
Abstract

HIV-1-positive individuals on successful antiretroviral therapy (ART) are reported to have higher rates of age-associated non-communicable comorbidities (AANCCs). HIV-associated immune dysfunction has been suggested to contribute to increased AANCC risk. Here we performed a cross-sectional immune phenotype analysis of T cells in ART-treated HIV-1-positive individuals with undetectable vireamia (HIV-positives) and HIV-1-negative individuals (HIV-negatives) over 45 years of age. In addition, two control groups were studied: HIV negative adults selected based on lifestyle and demographic factors (Co-morBidity in Relation to AIDS, or COBRA) and unselected age-matched donors from a blood bank. Despite long-term ART (median of 12.2 years), HIV-infected adults had lower CD4+ T-cell counts and higher CD8+ T-cell counts compared to well-matched HIV-negative COBRA participants. The proportion of CD38+HLA-DR+ and PD-1+ CD4+ T-cells was higher in HIV-positive cohort compared to the two HIV-negative cohorts. The proportion CD57+ and CD27-CD28- cells of both CD4+ and CD8+ T-cells in HIV-positives was higher compared to unselected adults (blood bank) as reported before but this difference was not apparent in comparison with well-matched HIV-negative COBRA participants. Multiple regression analysis showed that the presence of an increased proportion of terminally differentiated T cells was strongly associated with CMV infection. Compared to appropriately selected HIV-negative controls, HIV-positive individuals on ART with long-term suppressed viraemia exhibited incomplete immune recovery and increased immune activation/exhaustion. CMV infection rather than treated HIV infection appears to have more consistent effects on measures of terminal differentiation of T cells.

Many UC-authored scholarly publications are freely available on this site because of the UC's open access policies. Let us know how this access is important for you.

Main Content
For improved accessibility of PDF content, download the file to your device.
RESEARCH ARTICLE
Terminal differentiation of T cells is strongly
associated with CMV infection and increased
in HIV-positive individuals on ART and lifestyle
matched controls
Thijs Booiman1,2, Ferdinand W. Wit2,3,4, Arginell F. Girigorie1,2, Irma Maurer1, Davide De
Francesco5, Caroline A. Sabin5, Agnes M. Harskamp1, Maria Prins6, Claudio Franceschi7,
Steven G. Deeks8, Alan Winston9, Peter Reiss2,3,4, Neeltje A. Kootstra1*, on behalf of The
Co-morBidity in Relation to Aids (COBRA) Collaboration
1 Department of Experimental Immunology, Amsterdam Infection and Immunity Institute, Academic Medical
Center, University of Amsterdam, Amsterdam, The Netherlands, 2 Amsterdam Institute for Global Health and
Development, Amsterdam, The Netherlands, 3 Department of Global Health & Division of Infectious Disease,
Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands, 4 HIV Monitoring
Foundation, Amsterdam, The Netherlands, 5 Department of Infection and Population Health, University
College London, London, United Kingdom, 6 Public health service, Amsterdam, The Netherlands,
7 Department of Experimental, Diagnostic and Specialty Medicine, Alma Mater Studiorum Universita di
Bologna, Bologna, Italy, 8 Department of Medicine, University of California, San Francisco, California, United
States of America, 9 Imperial College of Science, Technology and Medicine, London, United Kingdom
¶ Members of The Co-morBidity in Relation to Aids (COBRA) Collaboration are provided in the
Acknowledgments.
* n.a.kootstra@amc.uva.nl
Abstract
HIV-1-positive individuals on successful antiretroviral therapy (ART) are reported to have
higher rates of age-associated non-communicable comorbidities (AANCCs). HIV-associ-
ated immune dysfunction has been suggested to contribute to increased AANCC risk. Here
we performed a cross-sectional immune phenotype analysis of T cells in ART-treated HIV-
1-positive individuals with undetectable vireamia (HIV-positives) and HIV-1-negative individ-
uals (HIV-negatives) over 45 years of age. In addition, two control groups were studied: HIV
negative adults selected based on lifestyle and demographic factors (Co-morBidity in Rela-
tion to AIDS, or COBRA) and unselected age-matched donors from a blood bank. Despite
long-term ART (median of 12.2 years), HIV-infected adults had lower CD4+ T-cell counts
and higher CD8+ T-cell counts compared to well-matched HIV-negative COBRA partici-
pants. The proportion of CD38+HLA-DR+ and PD-1+ CD4+ T-cells was higher in HIV-positive
cohort compared to the two HIV-negative cohorts. The proportion CD57+ and CD27−CD28−
cells of both CD4+ and CD8+ T-cells in HIV-positives was higher compared to unselected
adults (blood bank) as reported before but this difference was not apparent in comparison
with well-matched HIV-negative COBRA participants. Multiple regression analysis showed
that the presence of an increased proportion of terminally differentiated T cells was strongly
associated with CMV infection. Compared to appropriately selected HIV-negative controls,
HIV-positive individuals on ART with long-term suppressed viraemia exhibited incomplete
immune recovery and increased immune activation/exhaustion. CMV infection rather than
PLOS ONE | https://doi.org/10.1371/journal.pone.0183357 August 14, 2017 1 / 17
a1111111111
a1111111111
a1111111111
a1111111111
a1111111111
OPEN ACCESS
Citation: Booiman T, Wit FW, Girigorie AF, Maurer
I, De Francesco D, Sabin CA, et al. (2017) Terminal
differentiation of T cells is strongly associated with
CMV infection and increased in HIV-positive
individuals on ART and lifestyle matched controls.
PLoS ONE 12(8): e0183357. https://doi.org/
10.1371/journal.pone.0183357
Editor: Guido Poli, Universita Vita Salute San
Raffaele, ITALY
Received: February 21, 2017
Accepted: August 2, 2017
Published: August 14, 2017
Copyright: © 2017 Booiman et al. This is an open
access article distributed under the terms of the
Creative Commons Attribution License, which
permits unrestricted use, distribution, and
reproduction in any medium, provided the original
author and source are credited.
Data Availability Statement: All relevant data are
within the paper and its Supporting Information
files.
Funding: The COBRA project has received funding
from the European Union’s Seventh Framework
Programme for research, technological
development and demonstration under grant
agreement no 305522. The funders had no role in
study design, data collection and analysis, decision
to publish, or preparation of the manuscript.
treated HIV infection appears to have more consistent effects on measures of terminal dif-
ferentiation of T cells.
Introduction
Antiretroviral therapy (ART) for human immunodeficiency virus type 1 (HIV-1) infection has
dramatically reduced AIDS-associated morbidity and mortality [1–3]. However, HIV-1-posi-
tive individuals on successful ART are reported to have higher rates of age-associated noncom-
municable comorbidities (AANCCs) than the general population [3–7]. Several contributing
factors have been implicated, including ART toxicity, chronic immune activation, immune
dysfunction and a higher prevalence of traditional risk factors [8, 9]. Interestingly, immuno-
logical alterations observed during treated HIV-1 infection reflect those observed in the gen-
eral population during aging [7, 10–12]. These include high levels of soluble inflammatory and
coagulation related proteins, high levels of T cell activation, high levels of T cell exhaustion,
low levels of naïve T cells and an extensive proliferative history of CD8 T cells [13–18].
Together, these age-associated immunological alterations are referred to as immune senes-
cence [19, 20], although the precise definition and clinical significance of this syndrome
remains controversial [4, 21, 22].
The immune senescent phenotype was first reported in a group of elderly adults who pro-
gressed more rapidly [23]. Subsequent work suggested that chronic viral infections such as
cytomegalovirus (CMV), hepatitis B virus (HBV), hepatitis C virus (HCV) and HIV, contrib-
ute to the development of this phenotype [8, 13, 15, 24–27]. Indeed, CMV infection is also
associated with low CD4:CD8 ratios, increased systemic inflammation and increased expan-
sion of terminally differentiated and senescent T cells [13, 15, 28, 29]. CMV prevalence is
extremely high in HIV-positive individuals and therefore CMV infection may well contribute
to the immune senescent phenotype observed in HIV-positive individuals. Recently, we dem-
onstrated that levels of terminal differentiation of T cells and immune senescence did not dif-
fer between HIV-positive individuals on ART and HIV-negative controls with comparable
age, lifestyle and demographic characteristics [30], findings which contrasted with those from
other studies [13–16]. To increase our understanding of these findings, we analysed the effect
of HIV-1 and CMV infection on T cell activation, exhaustion and terminal differentiation of T
cells in HIV-1-positive individuals with suppressed viraemia on ART (HIV-positive), HIV-
1-negative individuals (HIV-negative) comparable regarding most lifestyle and demographic
factors derived from the Co-morBidity in Relation to AIDS (COBRA) cohort, and age-
matched unselected blood bank donors (referred in the tables as BBD). Notably, these blood
bank donors are at lower risk of acquiring blood borne infections than the general population
but often used as a control group for comparative studies.
Materials and methods
Subjects
Study subjects other than the bloodbank donors participated in an ongoing European Com-
mission-funded project known as COBRA (Co-morBidity in Relation to AIDS). COBRA is a
detailed, prospective evaluation of the burden of AANCC among 134 HIV-1-positive patients
on ART and 79 appropriately chosen non-infected controls who have comparable socio-demo-
graphic and behavioral (risk) factors. COBRA aims to provide a robust estimate of the effect of
treated HIV infection on the prevalence, incidence and age of onset of AANCC. Furthermore,
Terminally differentiated T-cells in HIV+ on ART
PLOS ONE | https://doi.org/10.1371/journal.pone.0183357 August 14, 2017 2 / 17
Competing interests: F. W. N. M. W. has received
travel grants from Gilead Sciences, ViiV Healthcare,
Boehringer Ingelheim, Abbvie, and Bristol-Myers
Squibb. A.W. has received honoraria or research
grants from or been a consultant or investigator in
clinical trials sponsored by Abbott, Boehringer
Ingelheim, Bristol-Myers Squibb, Gilead Sciences,
GlaxoSmithKline, Janssen-Cilag, Roche, Pfizer and
ViiV Healthcare. P. R., through his institution,
received independent scientific grant support from
Gilead Sciences, Janssen Pharmaceuticals, Merck,
Bristol-Myers Squibb, and ViiV Healthcare; served
on a scientific advisory board for Gilead Sciences
and a data safety monitoring committee for
Janssen Pharmaceuticals; and chaired a scientific
symposium by ViiV Healthcare, for which his
institution has received remuneration. The other
authors report no conflicts of interest exist.We
confirm that the statement does not alter our
adherence to PLOS ONE policies on sharing data
and materials.
COBRA aims to clarify the pathogenic mechanisms underlying this causative link, including
the possible induction of an inflammation-associated accelerated ageing phenotype. Exclusion
criteria were: age under 45 years, and self-reported current intravenous drug use (in the past
six months), daily use of recreational drugs (with the exception of cannabis), and excess alco-
hol intake (>48 units per week). All HIV-positive participants were required to be on ART
and to have had undetectable plasma HIV RNA (<50 copies/mL) for 12 months prior to
enrolment. Participants were recruited from two study sites: London and Amsterdam. For this
immunological study, 40 HIV-positive and 40 HIV-negative participants were randomly
selected with equal numbers in each of the following age-groups (45–50, 51–55, 56–60, 61–65,
66–70, and 70+), except for the oldest age category in which only a few individuals were avail-
able. Materials from blood bank donors were obtained from the Dutch national blood bank in
Amsterdam, the Netherlands (www.sanquin.nl). Blood bank donors (median: 58, IQR: 52.0–
65.0) were matched for age with the HIV-positive (Median: 58.5, IQR: 53.5–63.5) and HIV-
negative (median: 59.0, IQR: 53.0–64.5) COBRA participants and selected in such a way that
the different age categories in the COBRA, except for the category of 70+, were equally repre-
sented. Blood bank donors from the Netherlands are actively screened for HIV, Hepatitis B,
Hepatitis C, syphilis, and HTLV infection. Individuals aged above 70 years or individuals that
display high risk behavior for blood born infections are excluded from blood donation. Of
note, blood bank donors infected with HBV but who cleared the infection documented by an
anti-HBs antibody titer of at least 200 IU/L or blood bank donors that have been vaccinated
against HBV are not excluded from blood donation.
Ethics statement
This study has been conducted in accordance with the ethical principles set out in the declara-
tion of Helsinki and was approved by the institutional review board of the Academic Medical
Center (AMC) (NL 30802.018.09), the London (Stanmore) Research Ethics Committee (REC)
(13/LO/0584), and the Ethics Advisory Body of the Sanquin Blood Supply Foundation in
Amsterdam. Written informed consent was obtained from all participants.
T cell phenotyping and flow cytometry
Cryopreserved PBMC were thawed and cell viability was analysed by trypan blue staining and
for FACS analysis cell viability was required to be >75%. PBMC were stained with monoclonal
antibodies for 30min at 4˚C in the dark, to determine expression levels of different T-cell
surface molecules. T cell differentiation was defined as the proportion of naïve (CD45RA+
CD27+CCR7+), central memory (CD45RA−CCR7+CD27+), transitional memory (CD45RA−
CCR7−CD27+), effector memory (CD45RA−CCR7−CD27−), and terminally differentiated
effector memory (TEMRA; CD45RA+CCR7−CD27−) within the total CD4 or CD8 T cell popu-
lation. T cell activation was defined as the proportion of cells that were positive for both CD38
and HLA-DR within the total CD4 or CD8 T cell population. T cell exhaustion was defined as
the proportion of PD1 positive cells within the total CD4 or CD8 T cell population. Terminally
differentiated T cells were defined as proportion of CD57 positive cells within the total CD4 or
CD8 T cells population, the proportion of cells negative for both CD27 and CD28 within the
total CD4 or CD8 T-cells population, or the proportion of CD57 positive within the CD28−
CD4+ or CD28−CD8+ T cell populations. The following directly conjugated monoclonal anti-
bodies were used for cell surface marker staining: CD3 V500, CD4 PE-Cy7, HLA-DR Fitc,
CD38 PE, CD28 PerCP Cy5.5, CD45RA PE-Cy7, CD8 Pacific Blue, CD57 APC, CCR7 PE,
CD27 PerCP Cy5.5 (BD Biosiences, San Jose, CA, USA), CD27 APCeFluor780, CD4 APC
eFluor780, and PD-1 PE (eBioscience, San Diego, CA, USA). Fluorescence was measured with
Terminally differentiated T-cells in HIV+ on ART
PLOS ONE | https://doi.org/10.1371/journal.pone.0183357 August 14, 2017 3 / 17
the FACS Canto II (BD Biosciences). The proportion of cells expressing each marker were
determined using FlowJo 7.6 (TreeStar, Ashland, OR, USA). The gating strategy is displayed in
the supplementary information (S1 Fig).
CMV antibody titers
CMV specific IgG levels are believed to be a surrogate marker of CMV reactivation and con-
current immune response to control infection [31]. Thus, increased CMV total antibody and
high avidity titers indicate recurrent CMV antigen exposure, whereas CMV high avidity titers
are also indicative of increased antibody maturation. CMV total antibody titers and high
avidity antibody titers were measured by ELISA-VIDITEST anti-CMV-IgG and IgG avidity
(VIDIA, Praha, Czech republic) according to the manufacturer’s instruction. For quantifica-
tion a standard curve was prepared by serial dilution of plasma from a known CMV seroposi-
tive individual.
Statistical analysis
Differences in subject characteristics between HIV-positive, HIV-negative and blood bank
donors were assessed using Student’s T test, Mann-Whitney U test, or Pearson Chi-square
test, as appropriate. Associations of immunological markers with study group (HIV-positive,
HIV-negative, blood bank donors) and CMV serostatus were evaluated using multivariable
linear regression adjusted for age and gender. In the sub-group of participants who were CMV
seropositive, we then considered the association of each immunological marker with group
and i) CMV total IgG and ii) CMV high avidity IgG, with the same adjustments. Some out-
comes were transformed to attain normality as indicated in the table. Unstandardized coeffi-
cient (B) indicates the difference in the outcome (immunological markers) with respect to the
variables (study group, CMV infection), and are given in the table. Uncorrected p values are
given in the tables and the required p value for significance after Bonferroni adjustment for
multiple testing is indicated. Analyses were performed in IBM SPSS Statistics for Windows
v.23 (IBM, Armonk, NY, USA) and GraphPad Prims 6 (GraphPad, La Jolla, CA, USA).
Results
Baseline characteristics of COBRA participants and blood bank donors
HIV-positive individuals had a median (IQR) age of 58.5 (53.5–63.5) years and HIV-negative
controls had a median (IQR) age of 59 (53–63.5) years. The groups were representative of the
main COBRA cohort study (HIV-positive individuals: 93.3% male, 86.6% MSM, 12.0% of Afri-
can descent; HIV-negative controls: 92.4% male, 79.8% MSM, 2.6% of African descent). HIV-
positive individuals had been diagnosed with HIV for a mean (standard deviation) of 13.9
(4.8) years ago, had been on ART for a mean (standard deviation) of 12.2 (4.7) years and had
spent a median (IQR) of 8.0 (5.3–10.9) years with an undetectable plasma viral load (Table 1).
Whilst the percentage of males did not differ significantly between the HIV-positive and HIV-
negative COBRA participants, it was significantly lower in the group of blood bank donors.
HIV-positive and HIV-negative COBRA participants were more often (co-)infected with
CMV and HCV as compared to the blood bank donors, whereas HIV-positive COBRA partici-
pants were more often infected with CMV, HBV and HCV when compared to HIV-negative
COBRA participants (Table 1). HIV-positive COBRA participants, despite long-term suppres-
sion of HIV-replication by ART, exhibited incomplete CD4+ T-cell restoration and elevated
CD8+ T-cell counts compared to HIV-negative COBRA participants, as reflected by lower
CD4 counts, higher CD8 counts and an inverted CD4:CD8 T-cell ratio (Table 1).
Terminally differentiated T-cells in HIV+ on ART
PLOS ONE | https://doi.org/10.1371/journal.pone.0183357 August 14, 2017 4 / 17
T cell activation and exhaustion in COBRA participants and blood bank
donors
The percentages of activated (HLA-DR+CD38+) CD4 T cells and exhausted (PD1+) CD4 T
cells were higher in ART-treated HIV-positive COBRA participants compared to both cohorts
of HIV-negative participants (Table 2, Fig 1A and 1B). Interestingly, the percentage of PD1+
expressing CD4 T cells was higher in both the HIV-positive and HIV-negative COBRA partici-
pants when compared to the blood bank donors (Fig 1B and Table 2). The percentage of acti-
vated (HLA-DR+CD38+) CD8 T cells was higher in the HIV-positive COBRA participants but
not in the HIV-negative COBRA participants when compared to the blood bank donors (Fig
1C and Table 2). No differences were observed in the percentage of exhausted (PD1+) CD8 T
cells between the three groups (Fig 1D and Table 2).
T cell differentiation in COBRA participants and blood bank donors
In the CD4 compartment, the percentage of naïve cells (CD45RA+CD27+CCR7+) was lower in
the HIV-positive COBRA participants compared to the HIV-negative COBRA participants, but
no differences were observed in the percentage of central memory (CD45RA−CCR7+CD27+),
transitional memory (CD45RA−CCR7−CD27+), effector memory (CD45RA−CCR7−CD27−)
and TEMRA cells (CD45RA+CCR7−CD27−) (Table 2). When compared to the blood bank
Table 1. Baseline characteristics HIV-positive and HIV-negative COBRA participants and blood bank donors.
HIV-positive
n = 40
HIV-negative
n = 40
HIV-positive vs HIV-
negative
Blood bank
donors n = 35
HIV-positive vs
BBD
HIV-negative vs
BBD
n (%) or median
(IQR)
n (%) or median
(IQR)
p value1 n (%) or median
(IQR)
p value1 p value1
Age (Years) 58.5 (53.5–63.5) 59.0 (53.0–64.5) 0.9 58 (52.0–65.0) 0.5 0.5
Male sex 36 (90.0%) 37 (92.5%) 0.7 18 (51.4%) < .001 < .001
African descent 5 (12.5%) 1 (2.5%) 0.09 n.a.
MSM 32 (80.0%) 30 (75.0%) 0.8 n.a.
CMV 38 (95.0%) 31 (77.5%) 0.02 8 (22.9%) < .001 < .001
anti-CMV IgG 50.9 (23.5–108.6) 23.9 (13.8–87.8) 0.03 11.3 (10.2–16.8) 0.002 0.09
High avidity anti-CMV IgG 30.7 (13.0–57.0) 13.3 (8.2–39.7) 0.048 10.7 (10.0–13.2) 0.046 0.4
HBV 21 (52.5%) 7 (17.5%) 0.001 n.a.
Cleared 18 (45.0%) 7 (17.5%) 0.008 n.a.
Chronic 3 (7.5%) 0 (0%) 0.08 0 (0%) 0.1 n.d.
HCV (chronic and acute) 6 (15%) 0 (0%) 0.01 0 (0%) 0.02 n.d.
CD4 counts, cells/μl 589 (470–800) 961 (759–1233) < .001 n.a.
CD8 counts, cells/μl 762 (636–1029) 488 (364–621) < .001 n.a.
CD4:CD8 ratio 0.80 (0.61–1.13) 1.95 (1.33–2.83) < .001 n.a.
CD4 nadir, cells/μl 180 (60–180)
Years since HIV diagnosis 13.9 (9.1–18.7)
Years since ART 12.2 (7.9–16.9)
Years undetectable plasma viral
load (<200 c/ml)2
8.0 (5.3–10.9)
1 p value calculated using Student’s t-test, Mann-Whitney U test or Chi-Square test, or Wilcoxon’s rank-sum test where applicable.
2 The threshold was set at 200 c/ml to exclude incidental viral blips from the period in which plasma viral load was detectable.
Abbreviations: HIV, human immunodeficiency virus; BBD, blood bank donors n.a., not available; n.d., not determined; IQR, interquartile range; MSM, men
who have sex with men; CMV, Cytomegalovirus; HBV, hepatitis B virus; HCV, hepatitis C virus; ART, antiretroviral therapy.
https://doi.org/10.1371/journal.pone.0183357.t001
Terminally differentiated T-cells in HIV+ on ART
PLOS ONE | https://doi.org/10.1371/journal.pone.0183357 August 14, 2017 5 / 17
donors, we observed that HIV-positive and HIV-negative COBRA participants had a higher
percentage of effector memory cells (CD45RA−CCR7−CD27−), whereas an additional increase
in the percentage of TEMRA cells (CD45RA+CCR7−CD27−) was observed in the HIV-negative
COBRA participants (Table 2).
Within the CD8 compartment, the percentage of effector memory cells (CD45RA−CCR7−
CD27−) was higher in HIV-positive COBRA participants compared to the HIV-negative
COBRA participants, whereas no differences were observed in the other populations (Table 2).
When compared to the blood bank donors, HIV-positive and HIV-negative COBRA partici-
pants had a lower percentage of naïve CD8 cells (CD45RA+CD27+CCR7+). The percentage of
effector memory cells (CD45RA−CCR7−CD27−) was higher in HIV-positive, while the per-
centage of transitional memory (CD45RA−CCR7−CD27+) was lower and TEMRA cells
Table 2. T cell activation, exhaustion and differentiation in HIV-positive and HIV-negative COBRA participants and blood bank donors.
Markers HIV-positive median
(IQR)
HIV-negative median
(IQR)
HIV-pos vs HIV-
neg
p value1
BBD median (IQR) BBD vs HIV-
pos
p value1
BBD vs HIV-
neg
p value1
CD4+ T cells
% HLA-DR+CD38+ of CD4+ T cells2 2.58 (1.73–3.96) 1.56 (1.03–2.57) 0.001 2.09 (1.41–2.65) 0.06 0.06
% PD1+ of CD4+ T cells 2 6.77 (5.16–10.69) 6.2 (3.38–8.36) 0.015 4.2 (2.73–5.03) 2.0E-06 0.03
% CD57+ of CD4+ T cells 2 12.14 (6.32–18.43) 12.21 (7.01–19.31) 0.8 4.73 (2.91–8.47) 3.2E-04 0.001
% CD27−CD28− of CD4+ T cells 2 4.76 (1.14–8.49) 6.03 (1.46–12.25) 0.8 0.19 (0.05–0.57) 1.0E-06 3.0E-06
% CD57+ of CD4+CD28− T cells 58.88 (37.64–81.46) 79.59 (45.93–85.51) 0.07 32.23 (15.29–63.46) 0.015 2.9E-04
% CD45RA+CCR7+CD27+ of CD4+ T
cells 4
11.4 (4.9–20.01) 18.54 (9.62–29.43) 0.017 23.37 (13.06–40.8) 9.4E-05 0.1
% CD45RA−CCR7+CD27+ of CD4+ T
cells 4
16.6 (11.33–23.1) 15.99 (10.74–22.9) 0.5 19.4 (11.44–22.44) 0.5 0.4
% CD45RA−CCR7−CD27+ of CD4+ T
cells 2
23.99 (21.17–33.27) 23.87 (18.75–27.1) 0.3 27.16 (19.81–33.86) 0.8 0.1
% CD45RA−CCR7−CD27− of CD4+ T
cells 2
11.85 (7.57–24.88) 10.55 (6.57–18.93) 0.4 6.97 (3.87–9.37) .001 0.046
% CD45RA+CCR7−CD27− of CD4+ T
cells 2
0.93 (0.55–2.52) 2.05 (1.06–4.41) 0.1 0.74 (0.3–1.33) 0.2 0.039
CD8+ T cells
% HLA-DR+CD38+ of CD8+ T cells 2 7.46 (4.54–10.8) 5.6 (3.29–9.65) 0.2 4.05 (2.66–5.68) 9.7E-05 0.051
% PD1+ of CD8+ T cells 2 19.58 (14.06–23.91) 16.08 (11.56–24.02) 0.2 15.79 (12.42–20.41) 0.4 0.9
% CD57+ of CD8+ T cells 50.6 (36.81–57.2) 45.08 (35.57–57.44) 0.8 30.4 (14.91–42.4) .009 .031
% CD27−CD28− of CD8+ T cells 38.1 (24.8–46.4) 36.4 (22.55–50.05) 0.7 12.2 (5.56–20.7) 4.5E-06 3.4E-04
% CD57+ of CD8+CD28− T cells 3 75.75 (67.06–80.98) 79.31 (74.03–85.81) 0.045 61.4 (49.4–83.37) 0.6 0.037
% CD45RA+CCR7+CD27+ of CD8+ T
cells 4
6.31 (3.67–10.12) 8.36 (4.19–15.23) 0.08 18.46 (9.85–30.48) 1.3E-05 0.025
% CD45RA−CCR7+CD27+ of CD8+ T
cells 2
3.71 (2.81–5.69) 4.47 (1.87–7.61) 1.0 5.34 (3.08–8.79) 0.8 1.0
% CD45RA−CCR7−CD27+ of CD8+ T
cells 4
26.8 (19.12–33.19) 20.72 (15.22–27.11) 0.06 34.49 (29.81–42.38) 0.2 0.003
% CD45RA−CCR7−CD27− of CD8+ T
cells 2
19.18 (14.37–29.89) 12.9 (9.13–19.46) 0.006 9.23 (5.79–15.22) 0.001 0.3
% CD45RA+CCR7−CD27− of CD8+ T
cells 4
19.17 (12.96–24.8) 19.18 (11.26–36.78) 0.3 8.88 (5.44–20.4) 0.1 0.047
1 Multivariable linear regression, corrected for age and gender. Uncorrected p values are given. Bonferroni adjustment for multiple testing required a p value
of <0.0025 for significance.
2 LOG transformed to obtain normality.
3 acrsine transformed to obtain normality.
4 square root transformed to obtain normality
Abbreviations: HIV, human immunodeficiency virus; BBD, blood bank donors; IQR, interquartile range.
https://doi.org/10.1371/journal.pone.0183357.t002
Terminally differentiated T-cells in HIV+ on ART
PLOS ONE | https://doi.org/10.1371/journal.pone.0183357 August 14, 2017 6 / 17
(CD45RA+CCR7−CD27−) higher in HIV-negative COBRA participants as compared to the
blood bank donors (Table 2).
Percentage of terminally differentiated T cells is increased in COBRA
participants compared to blood bank donors
No significant differences in the percentages of terminally differentiated T cells between the
HIV-positive and HIV-negative COBRA participants were observed (Fig 2A and 2B and
Fig 1. CD4 and CD8 T cell activation and exhaustion in HIV-positive and HIV-negative COBRA participants, and
blood bank donors. (a) The percentage of activated (HLA-DR+CD38+) and (b) exhausted (PD-1+) CD4+ T cells within the
CD4+ T-cell population. (c) The percentage of activated (HLA-DR+CD38+) and (d) exhausted (PD-1+) T cells within the
CD8+ T-cell population. Significance was assessed with multivariable linear regression, corrected for age and gender.
HIV+, HIV-positive; HIV-, HIV-negative; BBD, blood bank donors.
https://doi.org/10.1371/journal.pone.0183357.g001
Terminally differentiated T-cells in HIV+ on ART
PLOS ONE | https://doi.org/10.1371/journal.pone.0183357 August 14, 2017 7 / 17
Table 2). Strikingly, a strong increase in the percentages of both terminally differentiated CD4
and CD8 T cells (CD57+ and CD27−/CD28−) was observed both when HIV-positive or HIV-
negative COBRA participants were compared to blood bank donors (Fig 2A and 2B and
Table 2). In addition, we analysed the percentage of CD57 expressing cells within the
CD28−CD8+ T-cells or CD28−CD4+ T cells as these cells have been shown to accumulate
upon chronic antigenic stimulation by viral pathogens [25]. The percentage of CD57 express-
ing cells within the CD28−CD4+ T cell population was not different between HIV-positive and
HIV-negative COBRA participants (Fig 2C and Table 2). However when compared to the
blood bank donors, the percentage of CD57 expressing cells within the CD28−CD4+ T cells
was higher in both the HIV-positive and HIV-negative COBRA participants (Fig 2C and
Table 2). The percentage of CD57 expressing cells within the CD28−CD8+ T cells was lower in
HIV-positive COBRA participants as compared to HIV-negative COBRA participants (Fig 2D
and Table 2). Blood bank donors have lower levels of CD57 expressing cells within the
CD28−CD8+ T cells when compared to HIV-negative COBRA participants, but not when
compared to HIV-positive COBRA participants (Table 2 and Fig 2D).
Fig 2. Terminally differentiated CD4 and CD8 T cells in HIV-positive and HIV-negative COBRA participants, and blood bank
donors. (a) The percentage of CD57+ and CD27−CD28− T cells within the CD4+ T cell population. (b) The percentage of CD57+ and
CD27−CD28− T cells within the CD8+ T cell population. (c) The percentage of CD57+ cells in the CD4+CD28− and (d) CD8+CD28−cell
population. Significance was assessed with multivariable linear regression, corrected for age and gender. HIV+, HIV-positive; HIV-, HIV-
negative; BBD, blood bank donors.
https://doi.org/10.1371/journal.pone.0183357.g002
Terminally differentiated T-cells in HIV+ on ART
PLOS ONE | https://doi.org/10.1371/journal.pone.0183357 August 14, 2017 8 / 17
HIV-1 and CMV infection are both associated with T cell activation,
exhaustion and terminal differentiation
Multivariable linear regression analysis was performed to determine whether HIV-1 infection
and CMV infection (CMV serostatus) were independently associated with T cell activation,
exhaustion, and terminal differentiation of T cells. after adjusting for age and gender. Within
the CD4 compartment, both T cell activation (HLA-DR+CD38+) and T cell exhaustion (PD1+)
were higher in HIV-positive participants compared to HIV-negative COBRA participants,
whereas T cell exhaustion was lower in blood bank donors as compared to HIV-negative
COBRA participants (Table 3). Within the CD8 compartment, HIV infection was associated
with higher T cell exhaustion while CMV infection was independently associated with lower
CD8 T cell exhaustion (PD1+) (Table 3).
When we analysed markers of terminally differentiated T cells (CD57+ and CD27−CD28−)
we observed that the percentage of CD27−CD28− cells within CD4+ T cells was lower in both
HIV-positive COBRA participants and blood bank donors when compared to HIV-negative
COBRA participants (Table 3). Moreover, a strong association between a higher proportion of
terminally differentiated (CD57+ and CD27−CD28−) CD4 and CD8 T cells and CMV infection
was observed (Table 3). The percentages of CD57 expressing cells within the CD28−CD8+ T-
cells and CD28−CD4+ T cells were lower in HIV-positive COBRA participants, while a positive
association with CMV infection was observed.
CMV IgG titers are associated with terminal differentiation of T cells and
CD8 T cell exhaustion
Anti-CMV IgG antibodies were positive in 95% of the HIV-positive COBRA participants,
77.5% of the HIV-negative COBRA participants, and 22.9% of the blood bank donors. HIV-
positive COBRA participants had higher levels of total and high avidity CMV-IgG antibody
titers as compared to both the HIV-negative COBRA participants and the blood bank donors
(Table 1), confirming previous observations [30, 32, 33]. Increased CMV IgG levels are sugges-
tive of increased stimulation by CMV antigens, which may further contribute to T cell activa-
tion, exhaustion and terminal differentiation. Total CMV IgG was associated with higher CD4
T cell activation (HLA-DR+CD38+), and terminally differentiated (CD57+ and CD27−CD28−)
CD4 and CD8 T cells in CMV-positive individuals irrespective of HIV-1 infection (Table 3).
In this model, CD4 T cell activation (HLA-DR+CD38+), CD4 T cell exhaustion (PD1+) and
CD8 T cell exhaustion (PD1+) were still higher in HIV-positive participants, whereas the per-
centage of CD4+CD27−CD28− T cells, the percentage of CD57 expressing cells within the
CD28−CD8+ T-cells and CD28−CD4+ T cells were lower in HIV-positive COBRA participants
when compared to HIV-negative COBRA participants (Table 3). Similarly, CMV high avidity
IgG levels were associated with higher CD4 T cell activation, terminally differentiated (CD57+
and CD27−CD28−) CD8 T cells, the percentage of CD57+ cells within CD8+CD28− cells, but
not terminally differentiated (CD57+ and CD27−CD28−) CD4 T cells (Table 3).
Discussion
Despite effective ART, HIV-positive individuals are reported to have higher rates of age-asso-
ciated non-communicable diseases and a shorter average life expectancy compared to unin-
fected persons of the same age [3, 5, 7]. The increased morbidity and mortality in the HIV-1
infected population may be the result of several contributing factors such as ART toxicity,
chronic immune activation and immune dysfunction as well as a higher prevalence of tradi-
tional risk factors for these AANCC [8, 9]. Here we analysed markers of T cell activation,
Terminally differentiated T-cells in HIV+ on ART
PLOS ONE | https://doi.org/10.1371/journal.pone.0183357 August 14, 2017 9 / 17
Table 3. Multivariable analyses of the association between HIV-1 infection, CMV serostatus, as well as CMV total and high avidity IgG titers, and T
cell activation, exhaustion and terminal differentiation in HIV-positive and HIV-negative COBRA participants and blood bank donors.
Complete population adjusted for
CMV serostatus1
CMV positive participants adjusted for
CMV Total IgG2
CMV positive participants adjusted
for CMV High Avidity IgG2
Activation and Exhaustion B (95% CI)5 p value B (95% CI) 5 p value B (95% CI) 5 p value
% HLA-DR+CD38+ of CD4+ T cells3 Group HIV-neg (ref)
HIV-pos 0.21 (0.10–0.32) 0.0003 0.18 (0.06–0.31) 0.005 0.19 (0.06–0.31) 0.003
BBD 0.10 (-0.038–0.24) 0.2 0.07 (-0.15–0.30) 0.5 0.07 (-0.15–0.29) 0.5
CMV 0.01 (-0.12–0.13) 0.9 0.001 (0.0003–0.002) 0.006 0.002 (0.0007–0.003) 0.003
% PD1+ of CD4+ T cells3 Group HIV-neg (ref)
HIV-pos 0.13 (0.02–0.24) 0.02 0.15 (0.04–0.26) 0.01 0.16 (0.04–0.27) 0.007
BBD -0.14 (-0.27–0.01) 0.04 -0.06 (-0.26–0.14) 0.6 -0.06 (-0.26–0.14) 0.5
CMV -0.002 (-0.12–0.12) 1.0 0.0003 (-0.0003–0.001) 0.3 0.0002 (-0.0009–0.001) 0.7
% HLA-DR+CD38+ of CD8+ T cells3 Group HIV-neg (ref)
HIV-pos 0.07 (-0.05–0.19) 0.3 0.03 (-0.11–0.17) 0.7 0.03 (-0.10–0.17) 0.6
BBD -0.11 (-0.26–0.03) 0.1 -0.23 (-0.47–0.01) 0.06 -0.23 (-0.48–0.009) 0.06
CMV 0.07 (-0.07–0.21) 0.3 0.00005 (-0.0003–0.001) 0.2 0.0008 (-0.0005–0.002) 0.2
% PD1+ of CD8+ T cells3 Group HIV-neg (ref)
HIV-pos 0.08 (-0.008–0.17) 0.07 0.11 (0.03–0.19) 0.008 0.11 (0.03–0.19) 0.007
BBD -0.07 (-0.18–0.04) 0.2 0.01 (-0.13–0.15) 0.9 0.01 (-0.13–0.15) 0.9
CMV -0.14 (-0.24–0.04) 0.006 -0.0006 (-0.0005–0.0004) 0.8 -0.0002 (-0.001–0.0006) 0.7
Terminal differentiation
% CD57+ of CD4+ T cells3 Group HIV-neg (ref)
HIV-pos -0.09 (-0.23–0.05) 0.2 -0.14 (-0.30–0.02) 0.08 -0.14 (-0.29–0.02) 0.09
BBD -0.13 (-0.30–0.04) 0.1 -0.16 (-0.44–0.13) 0.3 -0.16 (-0.45–0.12) 0.3
CMV 0.36 (0.20–0.51) 1.3E-05 0.001 (0.00003–0.002) 0.04 0.001 (-0.0001–0.003) 0.07
% CD27-CD28- of CD4+ T cells3 Group HIV-neg (ref)
HIV-pos -0.28 (-0.54–0.03) 0.03 -0.33 (-0.62–0.05) 0.02 -0.32 (-0.61–0.03) 0.03
BBD -0.35 (-0.66–0.04) 0.03 -0.49 (-1.00–0.03) 0.06 -0.49 (-1.01–0.02) 0.06
CMV 1.32 (1.03–1.61) 5.2E-15 0.002 (0.00005–0.003) 0.04 0.003 (-0.0002–0.006) 0.06
% CD57+ of CD8+ T cells Group HIV-neg (ref)
HIV-pos -1.89 (-8.07–4.28) 0.5 -3.77 (-10.52–2.99) 0.3 -3.63 (-10.25–2.99) 0.3
BBD -1.13 (-8.82–6.56) 0.7 -5.91 (-17.90–6.08) 0.3 -6.03 (-17.87–5.81) 0.3
CMV 16.02 (8.97–23.07) 1.6E-05 0.05 (0.016–0.09) 0.006 0.10 (0.04–0.17) 0.003
% CD27-CD28- of CD8+ T cells Group HIV-neg (ref)
HIV-pos -2.64 (-8.65–3.37) 0.4 -6.07 (-12.98–0.85) 0.09 -5.72 (-12.59–1.15) 0.1
BBD -5.22 (-12.70–2.27) 0.2 -15.21 (-27.49–2.94) 0.02 -15.40 (-27.68–3.12) 0.02
CMV 21.38 (14.51–28.23) 1.2E-08 0.05 (0.02–0.09) 0.007 0.09 (0.03–0.16) 0.008
% CD57+ of CD4+CD28- T cells Group HIV-neg (ref)
HIV-pos -17.67 (-27.73–7.61) 0.001 -18.31 (-29.31–7.32) 0.001 -18.67 (-29.45–7.88) 0.001
BBD -7.59 (-20.13–4.95) 0.2 -7.09 (-26.61–12.42) 0.5 -7.05 (-26.335–12.24) 0.5
CMV 39.74 (28.25–51.24) 4.5E-10 0.04 (-0.03–0.10) 0.2 0.10 (-0.01–0.20) 0.08
% CD57+ of CD8+CD28- T cells4 Group HIV-neg (ref)
HIV-pos -0.10 (-0.18–0.01) 0.03 -0.10 (-0.19–0.02) 0.02 -0.10 (-0.19–0.02) 0.02
BBD -0.05 (-0.16–0.06) 0.3 -0.07 (-0.23–0.08) 0.3 -0.07 (-0.23–0.08) 0.3
CMV 0.13 (0.03–0.23) 0.01 0.0004 (-0.0001–0.0009) 0.1 0.001 (0.00007–0.002) 0.04
1 Multivariable linear regression adjusted for age and gender in HIV-positive (n = 40), HIV-negative (n = 40) and BBD (n = 35). Uncorrected p values are
given. Bonferroni adjustment for multiple testing required a p value of <0.005 for significance.
2 Multivariable linear regression adjusted for age and gender in HIV-positive (n = 38), HIV-negative (n = 31) and BBD (n = 8) infected with CMV. Uncorrected
p values are given. Bonferroni adjustment for multiple testing required a p value of <0.005 for significance.
3 LOG transformed to obtain normality.
4 acrsine transformed to obtain normality.
5Unstandardized coefficient (B) indicates the difference in the outcome (immunological markers) with respect to the variables (study group, CMV infection).
For example, B is the increase in LOG transformed % of HLA-DR+CD38+ of CD4+ T cells in a HIV-positive individual as compared to a HIV-negative
participant.
Abbreviations: HIV, human immunodeficiency virus; BBD, blood bank donors; B, unstandardized regression coefficient; CI, confidence interval; CMV,
Cytomegalovirus; IgG, Immunoglobulin G
https://doi.org/10.1371/journal.pone.0183357.t003
Terminally differentiated T-cells in HIV+ on ART
PLOS ONE | https://doi.org/10.1371/journal.pone.0183357 August 14, 2017 10 / 17
exhaustion, and differentiation in a cohort of HIV-positive adults who were on apparently
effective ART and compared these to data from adults who were comparable for age, lifestyle
and demographic factors (COBRA) and a cohort age matched blood bank donors. HIV-posi-
tive COBRA participants showed incomplete immune recovery as reflected by lower CD4 T
cell counts and higher CD8 T cell counts than observed in either well-matched or just age-
matched uninfected controls. Furthermore, a higher percentage of activated and exhausted
CD4 cells, a lower percentage of CD4 naïve cells and higher percentage of CD8 effector mem-
ory cells were observed when compared to HIV-negative COBRA participants which is
broadly consistent with previous studies [30, 34–40]. In contrast, measures of terminally differ-
entiated T cells (CD57+ and CD27− CD28−), did not differ between the treated HIV-positive
and HIV-negative COBRA participants, confirming previous observations [17, 30], but con-
tradicting other studies [8, 13, 25, 27]. Collectively, these data provide strong support for the
concept that certain HIV-mediated immunologic perturbations (e.g., low CD4/CD8 ratio,
shift towards more differentiated memory cell types, high PD-1 expression) do indeed persist
indefinitely during ART, while other markers more typically associated with terminal differen-
tiation and senescence may normalize, at least compared to levels observed in well-matched
persons who lack HIV but have other risk factors, particularly CMV infection.
Strikingly, the percentages of activated CD8 T cells, exhausted CD4 T cells and terminally
differentiated CD4 and CD8 T cells were higher in both HIV-positive and HIV-negative
COBRA participants when compared to the general populations (e.g., the blood bank donors).
HIV-positive and negative COBRA participants are more often infected with CMV when com-
pared to the blood bank donors which may be due to the high number of men-who-have-sex-
with-men in the COBRA participants. Multivariable analysis showed that HIV-1 infection is
independently associated with CD4 T cell activation and T cell exhaustion but not with termi-
nally differentiated T cells (CD57+ and CD27−CD28−). CMV infection was strongly associated
with increased proportion of terminally differentiated T cells and may at least partially explain
the higher levels of terminally differentiated T cells in the HIV-positive and HIV-negative
COBRA participants. This is in line with previous publications in which CMV infection was
strongly associated with increased terminal differentiation of T cells and CD8 T cell exhaustion
[13, 15, 17, 28, 29]. However, no association of CMV infection with T cell activation was
observed which is in contrast with previous studies [41–43]. HIV-positive individuals in our
study are considered to be successfully treated, which is reflected in the high CD4 counts and
relatively high CD4:CD8 ratios. Furthermore, they have a relative low % of activated CD8 cells
(median 7,5% CD8+HLA-DR+CD38+) as compared to the other studies (15–20% CD8+HLA-
DR+CD38+) [41–43], and this might at least in part explain the differences between the studies.
We did however observe that higher CMV IgG titers were associated with higher CD4 T cell
activation in the CMV-positive participants.
HIV-positive COBRA participants had increased levels of CMV specific IgG when com-
pared to HIV-negative COBRA participants and blood bank donors, confirming previous
observations [30, 32, 33]. CMV specific IgG levels are considered a surrogate marker of CMV
reactivation and concurrent immune response to control infection, and high levels are associ-
ated with ageing and increased morbidity and mortality in both the general population and
HIV-positive individuals [30–33, 44–46]. Total CMV IgG was independently associated with
the presence of a higher proportion of terminally differentiated T cells and CD4 T cell activa-
tion, indicating that reactivation of CMV infection contributes substantially to terminal differ-
entiation of T cells and CD4 activation in treated HIV-positive individuals. This could suggest
that the high CMV prevalence combined with increased CMV reactivation may contribute to
the increased comorbidity burden that has been reported in treated HIV-positive individuals
[7]. Ageing and CMV infection are associated with terminal differentiation and proliferation
Terminally differentiated T-cells in HIV+ on ART
PLOS ONE | https://doi.org/10.1371/journal.pone.0183357 August 14, 2017 11 / 17
of CD8+CD28−CD57+ effector memory cells, however HIV-1 has been shown to inhibit
the terminal differentiation of these cells which is reflected in a decreased percentage of
CD28−CD8+ T-cells expressing CD57 [17, 18]. In line with these findings we observed that
HIV-1 infection was independently associated with a decrease in the percentage of CD57
expressing cells within the CD28−CD8+ T cells, whereas CMV infection was independently
associated with an increased percentage of these cells.
Cigarette smoking is known to have an impact on different immune functions, with both
proinflammatory and immune suppressive effects having been described [47, 48]. For exam-
ple, increased levels of markers of inflammation (CRP, IL-6, D-dimer, sCD14), T cells activa-
tion (% HLA-DR+CD38+ of CD4 and CD8 cells), and decreased T cell function (% PD1+ CD4
and CD8 T cells) have been associated with smoking in HIV-infected individuals as well as in
the general population [49–52]. In the present study, data about cigarette smoking was only
available for the COBRA participants (HIV-positive and HIV-negative), and in this group no
association between smoking and T cell activation, T cell exhaustion, and terminal differentia-
tion of CD4 and CD8 cells was observed (data not shown). However, we cannot exclude that
the limited number of individuals analysed and the lack of data about cigarette smoking in the
BBD group might explain why previously reported associations between cigarette smoking
and T cell activation and function could not be confirmed [49, 51].
Due to its observational nature, our study does suffer from several limitations. Firstly,
although we attempted to identify independent effects of HIV and CMV, CMV infection was
highly prevalent in both groups of COBRA participants, reflecting the high numbers of MSM
in each group (for example, only 2 of the 40 HIV-positive participants were CMV-negative).
Thus our ability to differentiate the independent effects of the two viruses is limited. For this
reason, analyses of CMV IgG may provide greater discriminative ability. Secondly, we cannot
rule out the possibility that the effects seen were a consequence of other unmeasured differ-
ences between the groups rather than HIV and CMV infection per se. Finally, the small num-
bers in some of our groups, and the low prevalence of coinfection with HBV and HCV meant
that we were unable to assess the effects of coinfection with these viruses.
In conclusion, HIV-positive individuals on ART with long-term suppressed viraemia
exhibited incomplete immune recovery and increased immune activation/exhaustion com-
pared to HIV-negative controls matched for age, lifestyle and demographic factors. However,
no evidence for increased immune senescence as determined by the level of terminally differ-
entiated T cells, was observed when HIV-positive individuals on suppressive ART were com-
pared to appropriately selected controls matched for age, lifestyle and demographic factors. If
only blood donors or perhaps someone poorly matched convenience sample had been used
as controls one would have falsely concluded that HIV was independently associated with
increased proportion of terminally differentiated T cells. This illustrates the importance of
recruiting and utilising appropriate control populations for such studies.
Supporting information
S1 Fig. Gating strategy flow cytometry. A. Gating of CD4+ and CD8+ T cells. B. T cell differ-
entiation was defined as the proportion of naïve (N; CD45RA+CD27+CCR7+), central memory
(CM; CD45RA−CCR7+CD27+), transitional memory (TM; CD45RA−CCR7−CD27+), effector
memory (EM; CD45RA−CCR7−CD27−), and terminally differentiated effector memory
(TEMRA; CD45RA+CCR7−CD27−) within the total CD4 or CD8 T cell population. C. T
cell activation was defined as the proportion of cells that were positive for both CD38 and
HLA-DR within the total CD4 or CD8 T cell population. T cell exhaustion was defined as the
proportion of PD1 positive cells within the total CD4 or CD8 T cell population. Terminally
Terminally differentiated T-cells in HIV+ on ART
PLOS ONE | https://doi.org/10.1371/journal.pone.0183357 August 14, 2017 12 / 17
differentiated T cells were defined as proportion of CD57 positive cells within the total CD4
or CD8 T cells population, the proportion of cells negative for both CD27 and CD28 within
the total CD4 or CD8 T-cells population, or the proportion of CD57 positive within the
CD28−CD4+ or CD28−CD8+ T cell population.
(PDF)
Acknowledgments
We would like to thank all the participants in the study for their time and effort. In addition
we would like to thank the COBRA Collaboration members at their respective sites:
Academisch Medisch Centrum, Universiteit van Amsterdam—Department of Global
Health and Amsterdam Institute for Global Health and Development (AIGHD): P. Reiss, F.W.N.
M. Wit, J. Schouten, K.W. Kooij, R.A. van Zoest, B.C. Elsenga, F.R. Janssen, M. Heidenrijk, W.
Zikkenheiner. Division of Infectious Diseases: M. van der Valk. Department of Experimental
Immunology: N.A. Kootstra, T. Booiman, A.M. Harskamp-Holwerda, I. Maurer, M.M. Mangas
Ruiz, A.F. Girigorie. Department of Medical Microbiology: J. Villaudy, E. Frankin, A. Pasternak,
B. Berkhout, T. van der Kuyl. Department of Neurology: P. Portegies, B.A. Schmand, G.J.
Geurtsen, J.A. ter Stege, M. Klein Twennaar. Department of Radiology: C.B.L.M. Majoie, M.W.
A. Caan, T. Su. Department of Cell Biology: K. Weijer. Division of Endocrinology and Metabo-
lism: P.H.L.T. Bisschop. Department of Experimental neuroendocrinology: A. Kalsbeek. Depart-
ment of Ophthalmology: M. Wezel. Department of Psychiatry: I. Visser, H.G. Ruhe´.
Alma Mater Studiorum Universita di Bologna—Department of Experimental, Diagnostic
and Specialty Medicine: C. Franceschi, P. Garagnani, C. Pirazzini, M. Capri, F. Dall’Olio, M.
Chiricolo, S. Salvioli.
Erasmus Universitair Medisch Centrum Rotterdam—Department of Genetics: J. Hoeij-
makers, J. Pothof.
GGD Amsterdam/Public Health Service Amsterdam—Cluster of Infectious Diseases,
research department: M. Prins, M. Martens, S. Moll, J. Berkel, M. Totte´, S. Kovalev.
Go¨teborgs Universitet—M. Gissle´n, D. Fuchs, H. Zetterberg.
Imperial College of Science, Technology and Medicine—Department of Medicine, Divi-
sion of Infectious Diseases: A. Winston, J. Underwood, L. McDonald, M. Stott, K. Legg, A. Lov-
ell, O. Erlwein, N. Doyle, C. Kingsley. Department of Medicine, Division of Brain Sciences, The
Computational, Cognitive & Clinical Neuroimaging Laboratory: D.J. Sharp, R. Leech, J.H. Cole.
Stichting HIV Monitoring—S. Zaheri, M.M.J. Hillebregt, Y.M.C. Ruijs, D.P. Benschop.
Stichting Katholieke Universiteit Nijmegen—D. Burger, M. de Graaff-Teulen.
Università degli studi di Modena e Reggio Emilia—Department of Medical and Surgical
Sciences for Children & Adults: G. Guaraldi.
Universita¨t Konstanz—Department of Biology: A. Bu¨rkle, T. Sindlinger, M. Moreno-Villa-
nueva, A. Keller.
University College London—Research Department of Infection and Population Health: C.
Sabin, D. de Francesco.
Vlaams Instituut voor Biotechnologie—Inflammation research center: C. Libert, S.
Dewaele.
Author Contributions
Conceptualization: Thijs Booiman, Caroline A. Sabin, Claudio Franceschi, Steven G. Deeks,
Alan Winston, Peter Reiss, Neeltje A. Kootstra.
Terminally differentiated T-cells in HIV+ on ART
PLOS ONE | https://doi.org/10.1371/journal.pone.0183357 August 14, 2017 13 / 17
Formal analysis: Thijs Booiman, Ferdinand W. Wit, Davide De Francesco, Caroline A. Sabin,
Neeltje A. Kootstra.
Funding acquisition: Maria Prins, Claudio Franceschi, Alan Winston, Peter Reiss, Neeltje A.
Kootstra.
Investigation: Thijs Booiman, Arginell F. Girigorie, Irma Maurer, Caroline A. Sabin, Agnes
M. Harskamp, Claudio Franceschi, Steven G. Deeks, Alan Winston, Neeltje A. Kootstra.
Methodology: Thijs Booiman, Ferdinand W. Wit, Arginell F. Girigorie, Irma Maurer, Davide
De Francesco, Caroline A. Sabin, Agnes M. Harskamp, Steven G. Deeks, Alan Winston,
Peter Reiss, Neeltje A. Kootstra.
Project administration: Agnes M. Harskamp, Alan Winston, Peter Reiss, Neeltje A. Kootstra.
Resources: Maria Prins, Claudio Franceschi, Alan Winston, Peter Reiss, Neeltje A. Kootstra.
Software: Thijs Booiman, Ferdinand W. Wit, Davide De Francesco.
Supervision: Caroline A. Sabin, Maria Prins, Steven G. Deeks, Alan Winston, Peter Reiss,
Neeltje A. Kootstra.
Validation: Thijs Booiman, Arginell F. Girigorie, Irma Maurer, Agnes M. Harskamp, Neeltje
A. Kootstra.
Visualization: Thijs Booiman, Neeltje A. Kootstra.
Writing – original draft: Thijs Booiman, Ferdinand W. Wit, Davide De Francesco, Caroline
A. Sabin, Steven G. Deeks, Peter Reiss, Neeltje A. Kootstra.
Writing – review & editing: Caroline A. Sabin, Peter Reiss, Neeltje A. Kootstra.
References
1. Wada N, Jacobson LP, Cohen M, French A, Phair J, Munoz A. Cause-specific life expectancies after 35
years of age for human immunodeficiency syndrome-infected and human immunodeficiency syn-
drome-negative individuals followed simultaneously in long-term cohort studies, 1984–2008. Am J Epi-
demiol. 2013; 177(2):116–25. https://doi.org/10.1093/aje/kws321 PMID: 23287403;
2. Weber R, Ruppik M, Rickenbach M, Spoerri A, Furrer H, Battegay M, et al. Decreasing mortality and
changing patterns of causes of death in the Swiss HIV Cohort Study. HIV Med. 2013; 14(4):195–207.
https://doi.org/10.1111/j.1468-1293.2012.01051.x PMID: 22998068.
3. Guaraldi G, Orlando G, Zona S, Menozzi M, Carli F, Garlassi E, et al. Premature age-related comorbidi-
ties among HIV-infected persons compared with the general population. Clin Infect Dis. 2011; 53
(11):1120–6. https://doi.org/10.1093/cid/cir627 PMID: 21998278.
4. Effros RB, Fletcher CV, Gebo K, Halter JB, Hazzard WR, Horne FM, et al. Aging and infectious dis-
eases: workshop on HIV infection and aging: what is known and future research directions. Clin Infect
Dis. 2008; 47(4):542–53. PMID: 18627268;
5. Hasse B, Ledergerber B, Furrer H, Battegay M, Hirschel B, Cavassini M, et al. Morbidity and aging in
HIV-infected persons: the Swiss HIV cohort study. Clin Infect Dis. 2011; 53(11):1130–9. https://doi.org/
10.1093/cid/cir626 PMID: 21998280.
6. Pathai S, Bajillan H, Landay AL, High KP. Is HIV a model of accelerated or accentuated aging? J Geron-
tol A Biol Sci Med Sci. 2014; 69(7):833–42. https://doi.org/10.1093/gerona/glt168 PMID: 24158766;
7. Schouten J, Wit FW, Stolte IG, Kootstra NA, van der Valk M, Geerlings SE, et al. Cross-sectional com-
parison of the prevalence of age-associated comorbidities and their risk factors between HIV-infected
and uninfected individuals: the AGEhIV cohort study. Clin Infect Dis. 2014; 59(12):1787–97. https://doi.
org/10.1093/cid/ciu701 PMID: 25182245.
8. Deeks SG. HIV infection, inflammation, immunosenescence, and aging. Annu Rev Med. 2011; 62:141–
55. https://doi.org/10.1146/annurev-med-042909-093756 PMID: 21090961;
9. Deeks SG, Tracy R, Douek DC. Systemic effects of inflammation on health during chronic HIV infection.
Immunity. 2013; 39(4):633–45. https://doi.org/10.1016/j.immuni.2013.10.001 PMID: 24138880;
Terminally differentiated T-cells in HIV+ on ART
PLOS ONE | https://doi.org/10.1371/journal.pone.0183357 August 14, 2017 14 / 17
10. Tenorio AR, Zheng Y, Bosch RJ, Krishnan S, Rodriguez B, Hunt PW, et al. Soluble markers of inflam-
mation and coagulation but not T-cell activation predict non-AIDS-defining morbid events during sup-
pressive antiretroviral treatment. J Infect Dis. 2014; 210(8):1248–59. https://doi.org/10.1093/infdis/
jiu254 PMID: 24795473;
11. McComsey GA, Kitch D, Sax PE, Tierney C, Jahed NC, Melbourne K, et al. Associations of inflamma-
tory markers with AIDS and non-AIDS clinical events after initiation of antiretroviral therapy: AIDS clini-
cal trials group A5224s, a substudy of ACTG A5202. J Acquir Immune Defic Syndr. 2014; 65(2):167–
74. https://doi.org/10.1097/01.qai.0000437171.00504.41 PMID: 24121755;
12. Karim R, Mack WJ, Kono N, Tien PC, Anastos K, Lazar J, et al. T-cell activation, both pre- and post-
HAART levels, correlates with carotid artery stiffness over 6.5 years among HIV-infected women in the
WIHS. J Acquir Immune Defic Syndr. 2014; 67(3):349–56. https://doi.org/10.1097/QAI.
0000000000000311 PMID: 25314253;
13. Appay V, Fastenackels S, Katlama C, Ait-Mohand H, Schneider L, Guihot A, et al. Old age and anti-
cytomegalovirus immunity are associated with altered T-cell reconstitution in HIV-1-infected patients.
AIDS. 2011; 25(15):1813–22. https://doi.org/10.1097/QAD.0b013e32834640e6 PMID: 21412126.
14. Appay V, Dunbar PR, Callan M, Klenerman P, Gillespie GM, Papagno L, et al. Memory CD8+ T cells
vary in differentiation phenotype in different persistent virus infections. Nat Med. 2002; 8(4):379–85.
https://doi.org/10.1038/nm0402-379 PMID: 11927944.
15. Kalayjian RC, Landay A, Pollard RB, Taub DD, Gross BH, Francis IR, et al. Age-related immune dys-
function in health and in human immunodeficiency virus (HIV) disease: association of age and HIV
infection with naive CD8+ cell depletion, reduced expression of CD28 on CD8+ cells, and reduced thy-
mic volumes. J Infect Dis. 2003; 187(12):1924–33. https://doi.org/10.1086/375372 PMID: 12792869.
16. Le Priol Y, Puthier D, Lecureuil C, Combadiere C, Debre P, Nguyen C, et al. High cytotoxic and specific
migratory potencies of senescent CD8+ CD57+ cells in HIV-infected and uninfected individuals. J
Immunol. 2006; 177(8):5145–54. PMID: 17015699.
17. Lee SA, Sinclair E, Hatano H, Hsue PY, Epling L, Hecht FM, et al. Impact of HIV on CD8+ T cell CD57
expression is distinct from that of CMV and aging. PLoS One. 2014; 9(2):e89444. https://doi.org/10.
1371/journal.pone.0089444 PMID: 24586783;
18. Lee SA, Sinclair E, Jain V, Huang Y, Epling L, Van Natta M, et al. Low proportions of CD28- CD8+ T
cells expressing CD57 can be reversed by early ART initiation and predict mortality in treated HIV infec-
tion. J Infect Dis. 2014; 210(3):374–82. https://doi.org/10.1093/infdis/jiu109 PMID: 24585893;
19. Franceschi C, Monti D, Barbieri D, Salvioli S, Grassilli E, Capri M, et al. Successful immunosenescence
and the remodelling of immune responses with ageing. Nephrol Dial Transplant. 1996; 11 Suppl 9:18–
25. PMID: 9050030.
20. Franceschi C, Bonafe M, Valensin S. Human immunosenescence: the prevailing of innate immunity,
the failing of clonotypic immunity, and the filling of immunological space. Vaccine. 2000; 18(16):1717–
20. PMID: 10689155.
21. Deeks SG, Phillips AN. HIV infection, antiretroviral treatment, ageing, and non-AIDS related morbidity.
BMJ. 2009; 338:a3172. https://doi.org/10.1136/bmj.a3172 PMID: 19171560.
22. Deeks SG. Immune dysfunction, inflammation, and accelerated aging in patients on antiretroviral ther-
apy. Top HIV Med. 2009; 17(4):118–23. PMID: 19890183.
23. Skiest DJ, Rubinstien E, Carley N, Gioiella L, Lyons R. The importance of comorbidity in HIV-infected
patients over 55: a retrospective case-control study. Am J Med. 1996; 101(6):605–11. https://doi.org/
10.1016/S0002-9343(96)00329-4 PMID: 9003107.
24. Naeger DM, Martin JN, Sinclair E, Hunt PW, Bangsberg DR, Hecht F, et al. Cytomegalovirus-specific T
cells persist at very high levels during long-term antiretroviral treatment of HIV disease. PLoS One.
2010; 5(1):e8886. https://doi.org/10.1371/journal.pone.0008886 PMID: 20126452;
25. Dock JN, Effros RB. Role of CD8 T Cell Replicative Senescence in Human Aging and in HIV-mediated
Immunosenescence. Aging Dis. 2011; 2(5):382–97. PMID: 22308228;
26. Grady BP, Nanlohy NM, van Baarle D. HCV monoinfection and HIV/HCV coinfection enhance T-cell
immune senescence in injecting drug users early during infection. Immun Ageing. 2016; 13:10. https://
doi.org/10.1186/s12979-016-0065-0 PMID: 27034702;
27. Brenchley JM, Karandikar NJ, Betts MR, Ambrozak DR, Hill BJ, Crotty LE, et al. Expression of CD57
defines replicative senescence and antigen-induced apoptotic death of CD8+ T cells. Blood. 2003; 101
(7):2711–20. https://doi.org/10.1182/blood-2002-07-2103 PMID: 12433688.
28. Freeman ML, Mudd JC, Shive CL, Younes SA, Panigrahi S, Sieg SF, et al. CD8 T-Cell Expansion and
Inflammation Linked to CMV Coinfection in ART-treated HIV Infection. Clin Infect Dis. 2016; 62(3):392–
6. https://doi.org/10.1093/cid/civ840 PMID: 26400999;
Terminally differentiated T-cells in HIV+ on ART
PLOS ONE | https://doi.org/10.1371/journal.pone.0183357 August 14, 2017 15 / 17
29. Tassiopoulos K, Landay A, Collier AC, Connick E, Deeks SG, Hunt P, et al. CD28-negative CD4+ and
CD8+ T cells in antiretroviral therapy-naive HIV-infected adults enrolled in adult clinical trials group stud-
ies. J Infect Dis. 2012; 205(11):1730–8. https://doi.org/10.1093/infdis/jis260 PMID: 22448010;
30. Cobos Jimenez V, Wit FW, Joerink M, Maurer I, Harskamp AM, Schouten J, et al. T-Cell Activation
Independently Associates With Immune Senescence in HIV-Infected Recipients of Long-term Antiretro-
viral Treatment. J Infect Dis. 2016. https://doi.org/10.1093/infdis/jiw146 PMID: 27073222.
31. Klenerman P, Oxenius A. T cell responses to cytomegalovirus. Nat Rev Immunol. 2016. https://doi.org/
10.1038/nri.2016.38 PMID: 27108521.
32. Parrinello CM, Sinclair E, Landay AL, Lurain N, Sharrett AR, Gange SJ, et al. Cytomegalovirus immuno-
globulin G antibody is associated with subclinical carotid artery disease among HIV-infected women. J
Infect Dis. 2012; 205(12):1788–96. https://doi.org/10.1093/infdis/jis276 PMID: 22492856;
33. Brunt SJ, Lee S, D’Orsogna L, Bundell C, Burrows S, Price P. The use of humoral responses as a
marker of CMV burden in HIV patients on ART requires consideration of T-cell recovery and persistent
B-cell activation. Dis Markers. 2014; 2014:947432. https://doi.org/10.1155/2014/947432 PMID:
25506120;
34. Cockerham LR, Siliciano JD, Sinclair E, O’Doherty U, Palmer S, Yukl SA, et al. CD4+ and CD8+ T cell
activation are associated with HIV DNA in resting CD4+ T cells. PLoS One. 2014; 9(10):e110731.
https://doi.org/10.1371/journal.pone.0110731 PMID: 25340755;
35. Chevalier MF, Petitjean G, Dunyach-Remy C, Didier C, Girard PM, Manea ME, et al. The Th17/Treg
ratio, IL-1RA and sCD14 levels in primary HIV infection predict the T-cell activation set point in the
absence of systemic microbial translocation. PLoS Pathog. 2013; 9(6):e1003453. https://doi.org/10.
1371/journal.ppat.1003453 PMID: 23818854;
36. Breton G, Chomont N, Takata H, Fromentin R, Ahlers J, Filali-Mouhim A, et al. Programmed death-1 is
a marker for abnormal distribution of naive/memory T cell subsets in HIV-1 infection. J Immunol. 2013;
191(5):2194–204. https://doi.org/10.4049/jimmunol.1200646 PMID: 23918986;
37. Said EA, Dupuy FP, Trautmann L, Zhang Y, Shi Y, El-Far M, et al. Programmed death-1-induced inter-
leukin-10 production by monocytes impairs CD4+ T cell activation during HIV infection. Nat Med. 2010;
16(4):452–9. https://doi.org/10.1038/nm.2106 PMID: 20208540;
38. Lim A, Tan D, Price P, Kamarulzaman A, Tan HY, James I, et al. Proportions of circulating T cells with a
regulatory cell phenotype increase with HIV-associated immune activation and remain high on antiretro-
viral therapy. AIDS. 2007; 21(12):1525–34. https://doi.org/10.1097/QAD.0b013e32825eab8b PMID:
17630546.
39. Piconi S, Trabattoni D, Gori A, Parisotto S, Magni C, Meraviglia P, et al. Immune activation, apoptosis,
and Treg activity are associated with persistently reduced CD4+ T-cell counts during antiretroviral ther-
apy. AIDS. 2010; 24(13):1991–2000. https://doi.org/10.1097/QAD.0b013e32833c93ce PMID:
20651586.
40. Weiss L, Piketty C, Assoumou L, Didier C, Caccavelli L, Donkova-Petrini V, et al. Relationship between
regulatory T cells and immune activation in human immunodeficiency virus-infected patients interrupt-
ing antiretroviral therapy. PLoS One. 2010; 5(7):e11659. https://doi.org/10.1371/journal.pone.0011659
PMID: 20657770;
41. Gianella S, Massanella M, Richman DD, Little SJ, Spina CA, Vargas MV, et al. Cytomegalovirus replica-
tion in semen is associated with higher levels of proviral HIV DNA and CD4+ T cell activation during anti-
retroviral treatment. J Virol. 2014; 88(14):7818–27. https://doi.org/10.1128/JVI.00831-14 PMID:
24789781;
42. Hunt PW, Martin JN, Sinclair E, Epling L, Teague J, Jacobson MA, et al. Valganciclovir reduces T cell
activation in HIV-infected individuals with incomplete CD4+ T cell recovery on antiretroviral therapy. J
Infect Dis. 2011; 203(10):1474–83. https://doi.org/10.1093/infdis/jir060 PMID: 21502083;
43. Wittkop L, Bitard J, Lazaro E, Neau D, Bonnet F, Mercie P, et al. Effect of cytomegalovirus-induced
immune response, self antigen-induced immune response, and microbial translocation on chronic
immune activation in successfully treated HIV type 1-infected patients: the ANRS CO3 Aquitaine
Cohort. J Infect Dis. 2013; 207(4):622–7. https://doi.org/10.1093/infdis/jis732 PMID: 23204178.
44. Roberts ET, Haan MN, Dowd JB, Aiello AE. Cytomegalovirus antibody levels, inflammation, and mortal-
ity among elderly Latinos over 9 years of follow-up. Am J Epidemiol. 2010; 172(4):363–71. https://doi.
org/10.1093/aje/kwq177 PMID: 20660122;
45. Gkrania-Klotsas E, Langenberg C, Sharp SJ, Luben R, Khaw KT, Wareham NJ. Higher immunoglobulin
G antibody levels against cytomegalovirus are associated with incident ischemic heart disease in the
population-based EPIC-Norfolk cohort. J Infect Dis. 2012; 206(12):1897–903. https://doi.org/10.1093/
infdis/jis620 PMID: 23045624.
Terminally differentiated T-cells in HIV+ on ART
PLOS ONE | https://doi.org/10.1371/journal.pone.0183357 August 14, 2017 16 / 17
46. Lichtner M, Cicconi P, Vita S, Cozzi-Lepri A, Galli M, Lo Caputo S, et al. Cytomegalovirus coinfection is
associated with an increased risk of severe non-AIDS-defining events in a large cohort of HIV-infected
patients. J Infect Dis. 2015; 211(2):178–86. https://doi.org/10.1093/infdis/jiu417 PMID: 25081936.
47. Holt PG. Immune and inflammatory function in cigarette smokers. Thorax. 1987; 42(4):241–9. PMID:
3303428;
48. Stampfli MR, Anderson GP. How cigarette smoke skews immune responses to promote infection, lung
disease and cancer. Nat Rev Immunol. 2009; 9(5):377–84. https://doi.org/10.1038/nri2530 PMID:
19330016.
49. de Heens GL, van der Velden U, Loos BG. Cigarette smoking enhances T cell activation and a Th2
immune response; an aspect of the pathophysiology in periodontal disease. Cytokine. 2009; 47(3):157–
61. https://doi.org/10.1016/j.cyto.2009.05.006 PMID: 19616447.
50. Kooij KW, Wit FW, Booiman T, van der Valk M, Schim van der Loeff MF, Kootstra NA, et al. Cigarette
Smoking and Inflammation, Monocyte Activation, and Coagulation in HIV-Infected Individuals Receiving
Antiretroviral Therapy, Compared With Uninfected Individuals. J Infect Dis. 2016; 214(12):1817–21.
https://doi.org/10.1093/infdis/jiw459 PMID: 27683822.
51. Valiathan R, Miguez MJ, Patel B, Arheart KL, Asthana D. Tobacco smoking increases immune activa-
tion and impairs T-cell function in HIV infected patients on antiretrovirals: a cross-sectional pilot study.
PLoS One. 2014; 9(5):e97698. https://doi.org/10.1371/journal.pone.0097698 PMID: 24842313;
52. Yanbaeva DG, Dentener MA, Creutzberg EC, Wesseling G, Wouters EF. Systemic effects of smoking.
Chest. 2007; 131(5):1557–66. https://doi.org/10.1378/chest.06-2179 PMID: 17494805.
Terminally differentiated T-cells in HIV+ on ART
PLOS ONE | https://doi.org/10.1371/journal.pone.0183357 August 14, 2017 17 / 17


Top