Current HIV Research, 2011, 9, 000-000 1
1570-162X/11 $58.00+.00 © 2011 Bentham Science Publishers Ltd.
HIV Infection-Related Premature Immunosenescence: High Rates of
Immune Exhaustion After Short Time of Infection
Sara Ferrando-Martínez1,2, Ezequiel Ruiz-Mateos1, María Concepción Romero-Sánchez1,3,
Mª Ángeles Muñoz-Fernández2, Pompeyo Viciana4, Miguel Genebat1 and Manuel Leal*,1
1Laboratory of Immunovirology, Biomedicine Institute of Seville (IBiS), Service of Infectious Diseases, Virgen del Rocío
University Hospital, Seville, Spain; 2Laboratory of Molecular Immuno-Biology, Gregorio Marañón University Hospital,
Madrid, Spain; 3Department of Clinical Biochemistry, IBiS/CSIC/University of Seville, Virgen del Rocío University
Hospital, Seville, Spain; 4Service of Infectious Diseases, Virgen del Rocio University Hospital, Seville, Spain
Abstract: Premature immunosenescence has been reported in different HIV scenarios. However, how premature is the
HIV-related immunosenescent phenotype is still unknown. Thus, the aim of this study was to analyze the
immunosenescent status of young viraemic naive HIV-infected individuals, with less than four years from infection. To
this end, replicative senescence, activation and proliferation T-cell levels were analyzed in chronically HIV-infected
young individuals and both, elderly and young healthy controls. We show that young HIV-infected viraemic patients, with
less than four years from infection, have early immune exhaustion leading to a premature immunosenescence comparable
to healthy people 40 years elder. In addition, memory T-cell subsets showed greater alterations than elder healthy controls
and, in patients with high viral loads, CD57 expression at the memory T-cell subsets was correlated with lower viral
increases but higher CD4 T-cell lost during follow up.
Keywords: HIV, immunosenescence, CD57, premature immunosenescence, HAART onset.
age-related changes of the immune system [1, 2] and usually
involves T-cell subset distribution changes and CD57-
expressing T-cell accumulations [3-5]. Cytomegalovirus
(CMV)-asymptomatic infection has been reported as the
driven force of T-cell aging . Alongside, a high
parallelism between CMV and HIV-infections, including
CMV-reactivation in HIV-infected patients , has been
found. Both processes, HIV and CMV-infection, induce
aberrant activation rates and continuous T-cell proliferation,
putting HIV infection forward as an appeal model of
premature immunosenescence . Premature immunosene-
scence has been associated to a faster AIDS progression:
during the first year under highly active antiretroviral
therapy (HAART), HIV-infected patients gain 35 CD4 T
cells less every 10 years of chronological age . An aged
HIV-infected population, where the percentage of subjects
older than 50 is continuously increasing [8-10], point out the
importance of the immunosenescence analysis in HIV-
Immunosenescence is a complex process that includes all
scenarios: acute HIV infection , HAART-treated
individuals [6, 7, 11], virological failure and structured
treatment interruptions , children  or patients with
low level of CD4 T-cell repopulation . When comparing
HIV-infected patients’ premature immunosenescence with
healthy aging, cellular phenotypes are similar in HAART-
Immunosenescence has been reported in different HIV
*Address correspondence to this author at the Laboratory of
Immunovirology, Service of Infectious Diseases, Virgen del Rocío
University Hospital, Avda. Manuel Siurot s/n, 41013 Seville, Spain;
Tel: +34 955 01 30 98; Fax: +34 955 01 32 92;
treated patients and 80 years-old HIV-negative individuals
despite good immune reconstitution and undetectable viral
loads [9, 15]. This result suggests that immune exhaustion
achieved before a successful HAART could not be reversed.
In addition, a frailty phenotype, similar to those used in
gerontology practice, has been reported in young HIV
patients naive to antiretroviral therapy [16, 17]. However,
when untreated viraemic patients are studied [18, 19], the
wide range of ages included confound premature
immunosenescence itself, making difficult the interpretation
of the results. In consequence, it will be important to study
the T-cell senescence level before HAART, since it seems to
be irreversible . Since young diagnosed-individuals have
now a long live expectancy thanks to HAART, determining
how early the immunosenescent phenotype appears could be
important to diminish the premature immunosenescent
burden by establishing the correct time for HAART onset.
with the chronological
the early immune exhaustion phenotype in young HIV-
infected subjects before their first antiretroviral therapy.
Thus, the aim of this study was to address the extent of
MATERIALS AND METHODS
eighteen consecutive HIV-infected individuals were selected
from the cohort of the Infectious Services Diseases Unit at
the Virgen del Rocio University Hospital, Seville, Spain.
Inclusion criteria were: 1) time from diagnose longer than
one year (chronic infection) but shorter than four years; 2)
CD4 T-cell counts over 350 cells/mm3 and 3) naive to
antiretroviral therapy. Individuals with an active infection
status, including Hepatitis C virus co-infection, were
excluded. When a previous HIV negative test was
documented within two years before diagnose (n = 14) time
Study subjects. From January 2008 to April 2008,
2 Current HIV Research, 2011, Vol. 9, No. 5 Ferrando-Martínez et al.
from infection was assessed as the half date between the
negative and the positive tests. In patients with no previous
HIV negative test time from infection was estimated based
on patient-reported high-risk exposure dates. Eight of the 18
patients had viral loads higher than 10000 HIV-RNA
copies/mL and were classified as the “High Viral Load
group”, while the other ten individuals, with viral loads
under 10000 HIV-RNA copies/mL, were the “Low Viral
Load group”. In November 2009, clinical records of all
patients were reviewed. CD4 T-cell counts and viral loads
were annotated in the last visit (if they were still naive) or
just before the HAART onset. Then, “CD4 T-cell count
difference” and “Viral Load difference” during the follow up
period (median time 1.4 years) were calculated.
as the Young Control group. Nineteen HIV-negative CD4
absolute counts-matched old donors, elder than 50, were
selected as the Elderly Control group. None of the study
subjects had received any treatment that could influence their
immune status and there were no clinical data of active
infections, including an HIV negative test.
Thirteen HIV-negative age-matched donors were selected
the study was approved by the Ethical Committee of the
All patients and controls signed an informed consent and
measured in fresh samples by quantitative PCR (COBAS
Ampliprep/COBAS Taqman HIV-1 test; Roche Molecular
Systems, Base Switzerland) according to the manufacturers’
HIV Viral Load determination. Plasma HIV-1 RNA was
determined in fresh whole blood using the Epics XL-MCL
(Beckman-Coulter Inc., California) flow cytometer, according
to the manufacturers’ instructions. CD4 and CD8 peripheral T-
cell subsets were positively selected from fresh PBMCs using
magnetic microbeads and MACS® Cell Separation Reagents
(Milteny Biotec, Germany) according to the manunfacturers’
instructions. CD4 and CD8 isolated T-cells were then separately
stained using anti-CD45RA FITC, anti-CD45RO PE-Cy7
(Becton Dickinson, San Jose, CA) and anti-CD57 FITC, anti-
CD38 PE, anti-CD45RA ECD, anti-HLADR ECD, anti-CD27
PE-Cy5 (Beckman Coulter, Florida, USA) monoclonal
antibodies. T-cell subsets were defined as follows: naive T-cells
CD45RA+CD27+; memory T-cells CD45RO+CD27+; effector
T-cells CD45RA+/-CD27-. The accuracy of these phenotypes
was recently reported (20). Proliferating T-cell levels were
determined by surface staining followed by intracellular
immunostaining with anti-Ki67 PE (Becton Dickinson, San
Jose CA) monoclonal antibody. Cells were permeabilized using
the Cytofix/Cytoperm, Perm/Wash and Cytoperm plus buffers
(BD Biosciences, USA).
Flow cytometry. CD4 and CD8 absolute counts were
median (interquartile range (IQR)) and categorical ones as
percentage. Pearson test was used to analyze correlations
between Gaussian variables and Spearman test was used to
analyze correlations between
Differences among categorical variables were analyzed using
the Chi square test. The Mann Whitney U-test was used to
analyze differences between continuous variables. Statistical
analysis was performed using the Statistical Package for the
Social Sciences software (SPSS 17.0, Chicago, Illinois, USA).
Statistical analysis. Continuous variables are expressed as
alterations similar to elderly controls. To determine whether
subset distribution changes observed in HIV-infected
patients were consistent with those changes described in
chronological immunosenescence, we compared young HIV-
infected individuals with age-matched young and CD4-
matched elderly healthy donors. Characteristics of the
different groups are shown in Table 1. When analyzing the
CD8 T-cell compartment, which is greater impaired in
chronological immunosenescence, the HIV-infected group
showed intermediate percentage of naive T-cells, statistically
diminished regarding to their age group, but unchanged
memory T-cell levels. Percentages of effector T-cells and
CD57 expression levels on total CD8 T-cells were
comparable to those showed by elderly controls and, in both
cases, significantly increased regarding the young group.
Young HIV-infected patients show CD8 T-cell
expression levels than the elderly group (Fig. 1A and Table
1) while activation and proliferation rates were higher at the
HIV-infected group than healthy controls, regardless of their
ages (Fig. 1B). In addition, memory CD8 T-cells showed the
highest levels of CD57 expression, even higher than those
showed by elderly people (Fig. 1C and Table 1), in
agreement with the accumulation of aberrant CD57-
expressing memory T-cells [18, 22]. Memory CD8 T-cells
also showed increased activation and proliferating levels
than the elder group (Fig. 1D). Interestingly, the two
individuals showing the highest activation levels were the
only two HIV-infected subjects over forty, possibly showing
an additive effect of HIV infection and higher ages.
Effector CD8 T-cells also showed similar CD57
cell alterations than elderly controls. As shown in Table 1,
naive CD4 T-cells were not diminished in HIV-infected
individuals regarding their age-matched counterparts.
Nevertheless, percentages of memory CD4 T-cells showed a
great drop whereas the percentage of effector CD4 T-cells
was increased, when compared with the young group. Unlike
CD8 T-cells, CD57 expression at the CD4 T-cell
compartment was higher at the HIV-infected group
compared to health control groups.
Young HIV-infected individuals show higher CD4 T-
also increased regarding both, young and elderly healthy
individuals (Fig. 2A and Table 1). Moreover, the effector
CD4 T-cell subset showed high activation and proliferation
rates (Fig. 2B), being the proliferating T-cell levels of HIV-
infected subjects even higher than those showed by the
elderly group. Similar to memory CD8 T-cells, HIV-infected
individuals showed higher CD57 expression in memory CD4
T-cells (Fig. 2C and Table 1) and proliferation levels (Fig.
2D) than all healthy controls, regardless of their ages.
Activation rates, however, were increased regarding at the
young group but remain similar to elderly activation rates
(Fig. 2D). It should be noted that the same two individuals
over 40 that showed increased activation rates of memory
CD8 T-cells also showed high activation levels at the
memory CD4 T-cell compartment.
CD57 expression at the effector CD4 T-cell subset was
with CD4 T-cell and viral load changes. We then analyzed
the relationship between CD57 expression levels on memory
CD57-expressing memory T-cell levels are associated
Early Immune Exhaustion of HIV+ Patients Current HIV Research, 2011, Vol. 9, No. 5 3
T-cells and both, CD4 and viral load evolution during the
follow up period in the high viral load group (n = 8). Despite
the low number of patients analyzed, we found that patients
with higher percentages of CD57 expression tend to have
higher CD4 T-cell lost but lower viral load increase during
the follow up (Fig. 3A). The same relationships were found
at the CD4 T-cell compartment (Fig. 3B).
exhaustion T-cell phenotype in young naive HIV-infected
individuals after short time of infection. Results show that
four years of untreated-HIV infection lead to a premature
immunosenescent phenotype similar to 70 years-old healthy
controls. In addition, our results suggest that CD57-
expressing T-cells could have a role on viral replication
control but are also correlated with higher CD4 T-cell lost.
In this study we provide evidence of an early immune
However, it is important to note that time from infection (rather
than time from diagnose) was analyzed. This fact, which is the
greatest strength of our study, make difficult to recruit large
cohorts. Therefore, despite the limited number of individuals,
the total amount is adequate to statistically support our
conclusion. With this design, we found that short infection times
A limitation of the study is the number of patients analyzed.
are enough to drive the immune exhaustion of the effector CD8
T-cell subset in HIV-infected subjects. The immunosenescent
status of 30 years old HIV-infected individuals was similar, or
even higher, than 70 years old HIV-negative controls’
immunosenescence. In addition, percentages of terminally
differentiated (CD57+) T-cells at the memory CD8
compartment were higher in HIV-infected subjects than in
elderly controls. On the other hand, while chronological
immunosenescence affects mostly to virus-specific CD8 T-cells,
HIV directly impairs CD4 T-cell numbers and function.
Consistently, CD57 expression in every CD4 T-cell subset was
higher in HIV-infected individuals than in healthy controls,
regardless of their ages. It should be note that the only two HIV-
infected individuals elder than forty showed the highest
activation rates in both, CD8 and CD4 memory T-cell subsets,
also suggesting that premature immunosenescent defects could
be boosted with age. Premature immunosenescence has been
reported in different HIV scenarios. However, the rapidity
which HIV infection can exhaust the immune system needed to
be analyzed. In this study we show that immune exhaustion
occurs in the first four years after infection. This early immune
senescence could be weighing down the T-cell recovery after
HAART, being a future burden when these individuals get
Baseline Characteristics and Immunological Status of the Different Groups
p(1 vs 2)?
p(1 vs 3)?
Log10 Viral Load?
Time from diagnose (years)?
Time of infection (years)*?
Time of follow up (years)?
CD8 T-Cell Compartment?
Naive CD8+ T-cells (%)?
Memory CD8+ T-cells (%)?
Effector CD8+ T-cells (%)?
CD8+CD57+ T-cells (%)?
Memory CD8+CD57+ T-cells (%)?
Effector CD8+CD57+ T-cells (%)?
CD4 T-Cell Compartment?
Naive CD4+ T-cells (%)?
Memory CD4+ T-cells (%)?
Effector CD4+ T-cells (%)?
CD4+CD57+ T-cells (%)?
Memory CD4+CD57+ T-cells (%)?
Effector CD4+CD57+ T-cells (%)?
Median [IQR]; p = Mann Whitney U-test; * n = 14.
4 Current HIV Research, 2011, Vol. 9, No. 5 Ferrando-Martínez et al.
Fig. (1). CD8 T-cell subset analysis. Effector CD8 T-cells: Percentages of A) CD57 expression and B) Activation (circles) and proliferation
(triangles) levels among the different groups. Black circles or triangles represent High Viral Load HIV+ patients. E = elderly. Y = young.
Memory CD8 T-cells: Percentages of C) CD57 expression and D) Activation (circles) and proliferation (triangles) levels in among the
different groups. Black circles or triangles represent High Viral Load HIV+ patients. Ages (years) of the two HIV out-layers are showed. E =
elderly. Y = young.
Fig. (2). CD4 T-cell subset analysis. Effector CD4 T-cells: Percentages of A) CD57 expression and B) Activation (circles) and proliferation
(triangles) among the different groups. Black circles or triangles represent High Viral Load HIV+ subjects. E = elderly Y = young. Memory
CD4 T-cells: Percentages of C) CD57 expression and D) Activation (circles) and proliferation (triangles) levels among the different groups.
Black circles or triangles represent High Viral Load HIV+ subjects. Age (years) of the two HIV out-layers are showed. E = elderly. Y =
Early Immune Exhaustion of HIV+ Patients Current HIV Research, 2011, Vol. 9, No. 5 5
due to HIV-related impairment of the CD27+ to CD27-
differentiation step [18, 21], was associated with higher CD4
T-cell loss in HIV-infected individuals with viral loads
higher than 10000 HIV-RNA copies/mL. Despite the low
number of patients analyzed (n = 8) our results interestingly
show that CD57-expressing memory T-cell accumulation
was also correlated with lower viral load increases during the
monitoring period. Our results suggest that CD57-expressing
exhaust T-cell accumulations, with high effector functions
[22, 23], can partially control viral replication but,
eventually, lead to an increased rate of CD4 T-cell death,
leading one to believe that both interpretations of CD57
function; effector capacity  and exhaustion-related
marker ; are partially true in the HIV-infection model.
The aberrant accumulation of memory CD57+ T-cells,
from infection less than 18 months were studied
retrospectively . A substantial proportion of these
subjects, who were asymptomatic and did not need CD4 T-
cell guide treatment introduction, (CD4 T-cell counts above
350 cells/uL) progressed to AIDS as fast as within three
years. This study point out the importance of defining new
markers that could anticipate the necessity of treatment in
high-risk patients. Since immunosenescence is closed related
to AIDS progression in old HIV-infected patients [8-10],
establishing HAART onset
immunosenescence is settled down could be a long-term
benefit for patients that had been young infected.
Interestingly, in a recent study, individuals with time
before the premature
exhaustion in young HIV-infected patients despite short
times of infection. Besides, CD57 T-cell accumulations at
the memory T-cell subsets correlate with lower viral load
increases but higher CD4 T-cell lost during the follow up.
Despite further research is necessary to balance the risk of
immunological progression due to the immunosencent status
with secondary effects of a HAART treatment, our results
suggest that early HAART onset could help to delay the
achievement of high immunological exhaustion rates in
Taken together, our results show high rates of immune
uninterested help. We are also thankful to Marien Gutiérrez
Authors want to thank all patients and volunteers for their
Sancho, Francisca Cano and Magdalena Rodríguez for their
priceless help with patients’ management. SFM and ERM
have grants from the Fondo de Investigaciones Sanitarias
(CD10/00382 and CP08/00172, respectively). MCRS was
supported by a Fondo de Investigación Sanitaria grant
PI06/0915. This study has been supported by Redes
Temáticas de Investigación en SIDA (ISCIII RETIC
RD06/0006/0021 and RD06/006/0035),
Excelencia, Consejería de Innovación, Ciencia y Empresa
(P10-CTS-06313) and Consejería de Salud, Servicio
Andaluz de Salud (PI0278/10).
Aw D, Silva AB, Palmer DB. Immunosenescence: emerging
challenges for an ageing population. Immunol 2007; 120: 435-46.
Gruver AL, Hudson LL, Sempowski GD. Immunosenescence of
aging. J Pathol 2007; 211: 144-56.
Junge S, Cloeckner-Gruissem B, Zufferey R, et al. Correlation
between thymic emigrants and CD31+ (PECAM-1) CD4+ T cells
in normal individuals during aging and in lymphopenic children.
Eur J Immunol 2007; 37: 3270-80.
Czesnikiewicz-Guzik M, Lee WW, Cui D, et al. T cell subset-
specific susceptibility to aging. Clin Immunol 2008; 127: 107-18.
Pawelec G, Akbar A, Caruso C, Effros R, Grubeck-Loebenstein B,
Wikby A. Is immunosenescence infectious? TRENDS Immunol
2004; 25: 406-10.
Papagno L, Spina CA, Marchant A, et al. Immune activation and
CD8+ T-cell differentiation towards senescence in HIV-1 infection.
PLoS Biology 2004; 2: E20.
Peres A. As time goes by… Would CD4+ T cells depletion induce
early immunosenescence in HIV infected patients? Med
Hypotheses 2010; 74: 202.
Kirk JB, Goetz MB. Human immunodeficiency virus in an aging
population, a complication of success. J Am Geriatr Soc 2009; 57:
Desai S, Landay A. Early immune senescence in HIV disease. Curr
HIV/AIDS Rep 2010; 7: 4-10.
Rickabaugh TM, Jamieson BD. A challenge for the future: aging
and HIV infection. Immunol Res 2010; 48:59-71
Naeger DM, Martin JN, Sinclair E, et al. Cytomegalovirus-specific
T cells persist at very high levels during long-term antiretroviral
treatment of HIV disease. PLoS ONE 2010; 5:e8886.
Huang KH, Loutfy MR, Tsoukas CM, Bernard MF. Immune
correlates of CD4 decline in HIV-infected patients experiencing
virologic failure before undergoing treatment interruption. BMC
Infectious Diseases 2008; 8: 59-65.
Resino S, Galan I, Bellon J, Navarro ML, Leon JA, Muñoz-
Fernandez MA. Characterizing the immune system after long-term
undetectable viral load in HIV-infected children. J Clin Immunol
2003; 23: 279-89.
Fig. (3). CD57 expressing memory T-cell subsets. Association between CD4 absolute T-cell (grey squares) and Viral Load (black
diamonds) changes and the percentage of basal expression in A) CD57-expressing Memory CD8 T-cells and B) CD57-expressing Memory
6 Current HIV Research, 2011, Vol. 9, No. 5 Ferrando-Martínez et al. Download full-text
Molina-Pinelo S, Vallejo A, Diaz L, et al. Premature
immunosenescence in HIV-infected patients on highly active
antirretroviral therapy with low-level CD4 T cell repopulation. J
Antimicrob Chemother 2009; 64: 579-588.
Desai SR, Usuga X, Martinson J, et al. Immune senescence
activation and abnormal T cell homeostasis despite effective
HAART, a hallmark of early aging in HIV. 16th Conference on
Retroviruses and Opportunistic Infections. Montreal, Canada,
Desquilbet L, Jacobson LP, Fried LP, et al. HIV-1 infection is
associated with an earlier occurrence of a phenotype related to
frailty. J Gerontol 2007; 62A: 1279-86.
Desquilbet L, Margolice JB, Fried LP, et al. Relationship between
a frailty related-phenotype and progressive deterioration of the
immune system in HIV-infected men. J Acquir Immune Defic
Syndr. 2009; 50: 299-306.
Hoji A, Connolly N, Buchanan W, Rinald CR Jr. CD27 and CD57
expression reveals atypical differentiation of HIV-1-specific
memory CD8+ T cells. Clin Vaccine Immunol 2007; 14: 74-80.
Day CI, Kaufmann DE, Kiepiela JA, et al. PD-1 expression on
HIV-specific T cells is associated with T-cell exhaustion and
disease progression. Nature 2006; 443: 350-354.
Ferrando-Martinez S, Ruiz-Mateos E, Leal M. CD27 and CCR7
expression on naive T cells, are both necessary? Immunol Lett
2010; 127: 157-8.
Appay V, van Lier RA, Sallusto F, Roederer M. Phenotype and
function of human T lymphocyte subsets: consensus and issues.
Cytometry A 2008; 73: 975-83.
Sadat-Sowti B, Debre P, Mollet L, et al. An inhibitor of cytotoxic
function produced by CD8+CD57+ T lymphocytes from patients
suffering from AIDS and immunosuppressed bone marrow
recipients. Eur J Immunol 1994; 24: 2882-8.
Barbour JD, Ndhlovu LC, Xuan Tan Q, et al. High CD8+ T cell
activation marks a less differentiated HIV-1 specific CD8+ T cell
response that is not altered by suppression of viral replication.
PLoS One 2009; 4: e4408.
Chattopadhyay PD, Betts MR, Price DA, et al. The cytolytic
enzymes granzyme A, granzyme B, and perforina: expression
patterns, cell distribution, and their relationship to cell maturity and
brigh CD57 expression. J Leukoc Biol 2009; 85: 88-97.
Brenchley JM, Karandikar NJ, Betts MR, et al. Expression of
CD57 defines replicative senescence and antigen-induced apoptotic
death of CD8 T cells. Blood 2003; 101: 2711-20.
Chattopadhyay PK, Roederer M. Good cell, bad cell: flow
cytometry reveals T-cell subsets important in HIV disease.
Cytometry part A 2010; 77A: 614-622.
Received: October 13, 2010
Revised: August 24, 2011 Accepted: August 26, 2011