Association of Differentiation State of CD4+ + T Cells and
Disease Progression in HIV-1 Perinatally Infected
Elizabeth R. Sharp1, Christian B. Willberg1, Peter J. Kuebler1, Jacob Abadi2, Glenn J. Fennelly2, Joanna
Dobroszycki2, Andrew A. Wiznia2, Michael G. Rosenberg2, Douglas F. Nixon1*
1Division of Experimental Medicine, Department of Medicine, University of California San Francisco, San Francisco, California, United States of America, 2Jacobi Medical
Center, Bronx, New York, United States of America
Background: In the USA, most HIV-1 infected children are on antiretroviral drug regimens, with many individuals surviving
through adolescence and into adulthood. The course of HIV-1 infection in these children is variable, and understudied.
Methodology/Principal Findings: We determined whether qualitative differences in immune cell subsets could explain a
slower disease course in long term survivors with no evidence of immune suppression (LTS-NS; CD4%$25%) compared to
those with severe immune suppression (LTS-SS; CD4%#15%). Subjects in the LTS-NS group had significantly higher
frequencies of naı ¨ve (CCR7+CD45RA+) and central memory (CCR7+CD45RA2) CD4+ T cells compared to LTS-SS subjects
(p=0.0005 and ,0.0001, respectively). Subjects in the rapid progressing group had significantly higher levels of CD4+ TEMRA
(CCR72CD45RA+) cells compared to slow progressing subjects (p,0.0001).
Conclusions/Significance: Rapid disease progression in vertical infection is associated with significantly higher levels of
CD4+ TEMRA(CCR72CD45RA+) cells.
Citation: Sharp ER, Willberg CB, Kuebler PJ, Abadi J, Fennelly GJ, et al. (2012) Association of Differentiation State of CD4+ T Cells and Disease Progression in HIV-1
Perinatally Infected Children. PLoS ONE 7(1): e29154. doi:10.1371/journal.pone.0029154
Editor: Landon Myer, University of Cape Town, South Africa
Received February 8, 2011; Accepted November 21, 2011; Published January 11, 2012
Copyright: ? 2012 Sharp 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.
Funding: Funding was provided by a grant from the National Institutes of Health AI060379. The funders had no role in study design, data collection and analysis,
decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: email@example.com
Various studies have sought to determine an association
between the control of HIV-1 viremia and magnitude of the
HIV-1 specific immune response [1,2,3,4,5]. The results have
been inconsistent. The qualitative characteristics of the HIV-1-
specific T cell response have become the focus of intense study and
it has been suggested that the inability of these responses to control
viremia is due to a failure of these cells to fully differentiate .
In contrast to other chronic viral infections such as CMV, HIV-
1 infection appears to result in a maturational block in the
generation of the HIV-1-specific T cell responses with skewing
toward an effector memory, TEM, phenotype . This seems to
result in an overall decrease in the frequency of fully differentiated
effector memory, TEMRA, cells [6,7]. We have previously shown
that the frequency and absolute numbers of CD8+ HIV-specific
TEMRAcells in early HIV-1 infection negatively correlate with the
future viral load set point . As CD4+ T cells are also known to
be important in the control of HIV-1 viremia[9,10,11,12,13,14,
15,16,17,18], we sought to determine whether alterations in CD4+
T cell subpopulations were associated with disease progression.
We chose to study a population of vertically infected children, and
categorized them into two progression groups based on CD4%
values using revised guidelines published by the CDC in
1994 , subjects with no immune suppression (LTS-NS;
CD4%$25%), and subjects with severe immune suppression
(LTS-SS; CD4%$15%). Surprisingly, we found striking differ-
ences in the differentiation phenotype of CD4+ T cells between
the two groups.
Subject Cohort Characteristics
We analyzed peripheral blood samples from 58 children and
adolescents with vertically acquired HIV-1. As described in
Materials and Methods, these subjects were divided into two
groups of immunological progression based on CDC guidelines.
The characteristics of both groups are described in Table 1.
As the children were categorized according to percentage CD4+
T cell count, it was not surprising to find a statistically significant
difference in the viral loads between the two groups. Of particular
note, allofthe patientshad some level of ongoing viral replication, as
none of them maintained consistently undetectable viral loads. The
LTS-NS group contained more African-Americans than the LTS-
SSgroup,butthisdid notreachsignificance.TheLTS-SSgroup was
slightly older than the LTS-NS group, but again this did not reach
significance. There were no significant differences in treatment
regimen or adherence levels between the two clinical groups.
PLoS ONE | www.plosone.org1January 2012 | Volume 7 | Issue 1 | e29154
Comparison of Differentiation Profiles of Bulk and HIV-1-
specific CD8+ T cells Between Progression Groups
We first characterized the HIV-1-specific CD8+ T cell
population in the two groups. We hypothesized that there would
be more fully differentiated CD8+ TEMRAcells in the LTS-NS
subjects compared to LTS-SS subjects, both in the total CD8+ T
cell population and in Gag-specific CD8+ T cells, as has been
observed from studies from adult HIV-1 infected cohorts [20,21].
We performed surface staining and intracellular cytokine staining
on 17 LTS-NS subjects and 15 LTS-SS subjects, stimulating
PBMC with single Gag peptides.
Surface staining of the total CD8+ T cell population revealed a
significantly higher frequency of naı ¨ve T cells (CCR7+ CD45RA+)
in LTS-NS subjects (p=0.0066). We observed a trend towards
higher levels of TEM(CCR72CD45RA2) cells in the LTS-SS
group although this was not significant (p=0.2). There was no
difference in the levels of TCM(CCR7+ CD45RA2) or TEMRA
(CCR72CD45RA+) cells between the two groups (Figure 1A). We
characterized epitope-specific CD8+ T cells for maturation
profiles using intracellular cytokine staining. No differences in
the maturational profiles of epitope-specific CD8+ T cells between
the two groups were observed (Figure 1B).
Striking Differences in CD4+ T cell Maturational Profiles
between Progression Groups
We next analyzed the characteristics of CD4+ T cells in these
subjects. CD4+ cells were defined as CD3+CD82 cells. In a
different panel with all three markers we verified that, on average,
93% of CD3+CD82 cells were CD4+. Remarkable differences in
the maturational profiles of CD4+ T cells between the two
progression groups were observed. As shown in Figure 2, subjects
in the LTS-NS groups had much higher frequencies of naı ¨ve and
central memory CD4+ T cells, which were highly statistically
significant (p=0.0005 and p,0.0001). There was no difference in
the levels of effector memory CD4+ T cells (p=0.984).
Interestingly, subjects in the LTS-SS group had significantly
higher levels of TEMRAcells than LTS-NS subjects, which was also
highly statistically significant (p,0.0001).
Additionally, the levels of TNAIVEcells and TCMcells showed a
strong positive correlation with CD4% (Spearman r=0.713 and
0.716, respectively, and p,0.0001 for both) and negatively with
LVL (Spearman r=20.421, p=0.018; and Spearman r=20.731,
p,0.0001, respectively) (Figure 3A–D). In contrast, the levels of
TEMRA cells were strongly negatively correlated with CD4%
(Spearman r=20.836, p,0.0001) and positively correlated with
LVL (Spearman r=0.0026, p=0.0026) (Figure 3G and H).
In order to further characterize these CD4+ T cells, levels of
CD57 expression were also measured. CD57 has been described a
marker of T cell senescence . CD57 expression on CD4+ T
cells from LTS-SS subjects was much higher than that seen on
CD4+ T cells from the LTS-NS group, (p=0.0025) (Figure 4A).
The frequency of CD4+ T cells expressing CD57 was also
negatively correlatedwith CD4%
p=0.001) and positively correlated with LVL (Spearman
r=0.41, p=0.02) (Figure 4B and C).
Several recent studies have suggested that qualitative charac-
teristics of the HIV-1-specific T cell are associated with protection,
viral control, and rate of disease progression [8,20,21,23,24]. To
our knowledge this is the first study to compare the qualitative
characteristics of total CD8+ and CD4+ as well as HIV-specific T
cell responses in perinatally infected children with different levels
of disease progression, with a significant finding relating specific
CD4+ T cell profiles with disease progression rate. Moreover,
unlike previous studies which have focused on individuals of
Northern European descent, this work concentrated primarily on
African Americans and Hispanics.
The children in this study, all older than 10 years of age and
thus considered long-term survivors (LTS), were categorized,
based on CD4+ T cell percentage levels, into those with no
immune suppression (LTS-NS) and those with severe immune
suppression (LTS-SS), based on previously published CDC
guidelines . We observed a highly significant increase in the
frequency of naı ¨ve CD8+ T cells (TNAIVE) in the LTS-NS subjects
(p=0.0066), compared to the LTS-SS subjects, but no differences
in any other CD8+ T cell subsets. The differentiation profiles of
Gag-specific CD8+ T cells were similar between the progression
The most striking finding of this study was the CD4+ T cell
differentiation profiles between the two progression groups.
Subjects in the LTS-NS group had significantly higher levels of
naı ¨ve T cells (TNAIVE) and central memory (TCM) CD4+ T cells
than LTS-SS children (=0.0005 and p,0.0001, respectively). In
contrast, children in the LTS-SS group had significantly higher
levels of effector memory RA+ (TEMRA) cells (p,0.0001). These
data suggest that disease progression in these vertically infected
younger patients is different than adults and is associated with a
shift towards greater TEMRAcell numbers.
The shift towards greater TEMRA cells could represent a
disproportionate loss of certain T cell subsets in LTS-SS subjects
consistent with published data. A recent study showed that CD4+
cells with the TEMRAphenotype (CCR72CD45RA+) were more
prevalent in HIV-1-infected individuals than in uninfected
controls . They described that these cells were resistant to in
vitro infection by CCR5-tropic strains of HIV-1, despite robust
expression of CCR5. Loss of Naı ¨ve T cells was also observed in a
study of HIV infected infants. In this study, there was an
association between rapid disease progression and decreases in
naı ¨ve cells but with little effect on memory cells. The loss of naı ¨ve
cells appeared to be mediated through thymic dysfunction [26,27].
Our cohort consists of long term survivors of vertically acquired
Table 1. Patient cohort characteristics.
Immunological CategoryNCD4% LVLa
No immune suppression - LTS-NS (CD4% $25)3029.8% (27.1; 34.8)3.65 (2.85; 4.14)13.8 (10.9; 16.6)M=15 F=15H=9 AA=20
No immune suppression - LTS-NS (CD4%,15)288.25% (5.5; 11.5)4.79 (4.37; 5.11)15.6 (12.5; 18.1)M=11 F=17 H=13 AA=13
TOTAL58 24.5% (8.25; 31.0)4.28 (3.61; 4.81)14.3 (11.6; 17.52)M=26 F=32 H=22 AA=33
aLog viral load.
bH=Hispanic; AA=African American.
CD4+ Temra Cells in Pediatric HIV-1 Infection
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HIV-1 infection who were infected perinatally. The loss of naı ¨ve
and central memory cells and increased frequency of TEMRAcells
could therefore be due to a combination of a greater susceptibility
to HIV-1 related cell death, together with thymic dysfunction.
Future studies would need to address the relative contributions of
these conditions to the observations.
The greater susceptibility of naı ¨ve and central memory T cells
to loss could potentially be due to effects of chronic immune
stimulation from HIV-1. Additional studies would also benefit
from analyses looking at cell surface activation markers such as
CD38 or HLA-DR. The loss of naı ¨ve and central memory T cells,
with the accumulation of TEMRA cells, suggests altered T cell
maturation kinetics or a change in TEMRAlifespan in progressing
subjects. We observed a greater frequency of CD4+CD57+ cells in
the LTS-SS group that correlated negatively with CD4% and
positively with viremia. CD57 is a marker of senescence. The
relationship of the increased CD57+ frequency with other markers
of disease progression corroborates the proposed detrimental effect
of maturation. This suggests an inability of homeostatic mecha-
nisms to maintain the appropriate proportion of T cell phenotypes
necessary for HIV-1 control in progressing subjects and could be
related to the accumulation of TEMRAcells.
This study suggests that T cell maturation patterns are
significantly different in perinatally HIV-1-infected children with
Figure 1. Comparison of differentiation profiles of total CD8+ + T cells between progression groups (A) and Comparison of
differentiation profiles of Gag-specific effector CD8+ + T cells between progression groups (B). We used expression of CCR7 and CD45RA
to categorize CD8+ T cells into one of four differentiation phenotypes. Filled circles (N) represent LTS-SS patients and empty circles (#) represent
LTS-NS patients. The line in each column represents the median and the differentiation phenotype is beneath each column.
CD4+ Temra Cells in Pediatric HIV-1 Infection
PLoS ONE | www.plosone.org3January 2012 | Volume 7 | Issue 1 | e29154
different levels of disease progression, but there are some caveats
to be considered. The relatively small number of subjects in the
study precludes us from applying these findings to a general
pediatric population. The subjects were not all on the same
antiretroviral treatment regimens. However, we attempted to
minimize these concerns by choosing subjects for the study who: 1)
did not receive HAART in the first two years of life; 2) had some
levels of ongoing viral replication; and 3) were ARV-experienced,
except for two patients. Another equalizing factor is that both
groups, as a whole, had generally similar treatment adherence
rates. We also cannot be completely confident that the staining
protocol does not potentially alter the pattern of staining of CD4+
T cells, which would be avoided by staining antigen specific cells
with tetramers. However, few class II tetramers are now available
for such studies.
We observed an extremely strong correlation between increased
CD4+ TEMRAcells and more severe immunological suppression.
Although this suggests that these cells are accumulating during
progressive infection, more research into this area is needed. The
previously observed resistance of these terminally differentiated
cells to R5 tropic strains of HIV-1 is intriguing and may be part of
the explanation why these patients are long-term survivors despite
persistently low CD4+ T cell levels. These findings could be of
importance to the field of pediatric HIV-1 immunology as well as
the larger field of HIV-1 vaccine design.
Materials and Methods
The research involving human participants reported in this
study was approved by the University of California San Francisco
(UCSF) and Albert Einstein College of Medicine (AECOM)
institutional review boards IRB, with the approval number
H11613–19149. Informed written consent was obtained for all
subjects. All clinical investigation were conducted according to the
principles expressed in the Declaration of Helsinki.
Patient Sample Characteristics
All subjects attended the Pediatric HIV clinic at Jacobi Medical
Center in the Bronx, NY. The vast majority of attendees of the
Jacobi Pediatric HIV Clinic are either African-American or
Hispanic. Stored samples from these patients were selected for this
study based on clinical characteristics that allowed them to be
classified according to previously published CDC guidelines ,
as explained below. Heparinized whole-blood samples were
obtained from 58 subjects after informed consent, based on local
Institutional Review Board-approved protocols. Plasma HIV-1
RNA was measured with the Amplicor HIV-1 Monitor with a
lower limit of quantification at 50 copies RNA/ml (Roche
Diagnostic Systems, Branchburg, NJ).
All subjects were perinatally infected and over 10 years of age,
and can thus be defined as long-term survivors (LTS). Since all of
the patients were born in an era prior to the availability of
pediatric HAART (1996 or before), none of the subjects received
potent, suppressive combination therapy during the first two years
of life. All of the patients, with the exception of 2 children, were
either taking antiretrovirals (ARVs) or were ARV-experienced, at
the time of sampling. Additionally, all subjects were viremic at
time of sampling and most had variable treatment adherence rates.
There were no significant differences in treatment regimen or
adherence levels between the two clinical groups.
The general consensus in the pediatric HIV field is that the
CD4% value is the most valuable marker of disease progression
and is used by the CDC to classify levels of disease status in HIV
infected children . In this study, patients were categorized into
Figure 2. Comparison of differentiation profiles of total CD4 T cells between progression groups. We used expression of CCR7 and
CD45RA to categorize CD4 T cells into one of four differentiation phenotypes. Filled circles (N) represent LTS-SS patients and empty circles (#)
represent LTS-NS patients. The line in each column represents the median and the differentiation phenotype is beneath each column.
CD4+ Temra Cells in Pediatric HIV-1 Infection
PLoS ONE | www.plosone.org4 January 2012 | Volume 7 | Issue 1 | e29154
two groups based on CD4% values, according to CDC guidelines
. Subjects with a sustained CD4%$25% were considered
LTS with No Evidence of Immune Suppression (LTS-NS). Those
with a sustained CD4%#15% were considered LTS with Severe
Immune Suppression (LTS-SS).
Patient histories were used to categorizing patients. Only
patients that had always possessed CD4% levels very near or
above 25% were considered as an LTS-NS subject. Patients that
possessed CD4% levels very near or below 15% for several clinic
visits were grouped as an LTS-SS subject. A window was
delineated for each patient within which samples were assayed
immunologically. We chose a period of time in which samples
were available and the patient was as clinically stable as possible,
as defined by CD4% and viral loads. We acknowledge that a range
Figure 3. Correlations between frequency of CD4 T cell subsets and clinical characteristics. Log viral load (LVL) versus frequency of CD4 T
cell subsets are shown in A, C, E, and G. CD4% (of total white blood cells) versus frequency of CD4 T cell subsets are shown in B, D, F, and H. The CD4
T cell subset analyzed is at the beginning of each row.
CD4+ Temra Cells in Pediatric HIV-1 Infection
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of immunosuppresion exists within each category, but we have
given an overall classification of ‘‘no evidence’’ and ‘‘severe’’ to
help distinguish the groups.
Multi-parameter flow cytometry
Single peptides, comprising the HXB2 sequence of Gag (AIDS
Research and Reference Reagent Program, NIH), previously
identified as containing targeted epitopes were used as the
antigenic stimulus (Sharp et al., in preparation). The negative
control was media alone and the positive control was Staphylo-
coccal Enterotoxin B (SEB). We also stimulated cultures with a
peptide pool consisting of well-identified HLA class I restricted
CMV, EBV and Influenza epitopes (CEF) in patients that had
previously demonstrated reactivity.
Briefly, cryopreserved PBMC were thawed and cells were
stimulated for one hour with either: media alone, antigen, or
positive control. The antibody, CD107 a/b-PECy5 was added
with stimulation. Brefeldin A (Sigma-Aldrich, St. Louis, MI, USA)
was then added at a concentration of 5 mg/ml and cells incubated
overnight. The next day, PBMC were washed and stained with
antibodies, in combination, against CD4-Alexa700, CD8-Pacific
Blue, CD45RA-biotin, CCR7-PECy7, CD57-PECy5, and a live/
dead marker emitting in the aqua wavelength for 20 minutes at
4uC. Cells were washed twice and stained with the secondary
antibody streptavidin-Qdot655 for 20 minutes at 4uC. Cells were
then washed and fixed in 2% paraformaldehyde. The cells were
permeabilized using FACS Perm solution (BD Biosciences),
washed and stained using antibodies, in combination, against
CD3-ECD and IFN-c-APC for 30 minutes at 25uC. Following
staining, the cells were washed, fixed in 1% paraformaldehyde,
and collected on a BD LSR-II using FACS DIVA software (BD
Biosciences). Data was analyzed using FlowJo (TreeStar).
In all analyses a forward scatter (FSC)-height versus FSC-area
plot to exclude all cell conjugates was used. Dead cells were then
excluded by only gating on cells negative for the live/dead marker.
A FSC-area vs. side scatter (SSC)-area plot was used to define the
lymphocyte gate. T cells were selected by gating on CD3+
lymphocytes, followed by selection of CD8+ cells by gating on
CD3+CD8+ cells. CD4+ cells were defined as CD3+CD82 cells.
In panels that contained CD3, CD4, and CD8 antibodies, we
verified that, on average, 93% of CD3+CD82 cells were CD4+.
IFN-c+ cells were defined using an APC ‘‘fluorescence minus one’’
(FMO) sample. Quadrant gates were set for expression of CCR7
and CD45RA by using a QDot655 FMO and a PECy7 FMO.
IFN-c+ cells were further analyzed for expression of T-cell
memory markers in a CCR7 versus CD45RA.
A median (interquartile range) was used as a measure of central
tendency for continuous variables. We employed the Mann-
Whitney two-tailed t-test for all simple comparisons between two
groups. The Spearman Rank correlation test and linear regression
Figure 4. Association between CD57 expression on CD4 T cells and disease progression. A. Comparison of CD57 expression on CD4 T
cells between progression groups. B. Correlation between log viral load (LVL) and CD57 expression on CD4 T cells. C. Correlation between CD4% and
expression of CD57 on CD4 T cells.
CD4+ Temra Cells in Pediatric HIV-1 Infection
PLoS ONE | www.plosone.org6January 2012 | Volume 7 | Issue 1 | e29154
analyses were used to explore associations between 2 continuous Download full-text
variables. Differences between categorical data were calculated
using Fisher’s exact test. We considered a p-value of ,0.05
significant. All statistical analyses were performed using the
GraphPad Prism 4.03 software package (La Jolla, CA).
We thank the patients and their families for participating in this study.
Conceived and designed the experiments: ERS CBW AAW MGR DFN.
Performed the experiments: ERS CBW. Analyzed the data: ERS CBW
PJK MGR DFN. Contributed reagents/materials/analysis tools: ERS
CBW PJK JA GJF JD AAW MGR DFN. Wrote the paper: ERS CBW
PJK AAW MGR DFN.
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