HIV infection-associated immune activation occurs by
two distinct pathways that differentially affect CD4
and CD8 T cells
Marta Catalfamoa,1, Michele Di Masciob, Zonghui Hub, Sharat Srinivasulac, Vishakha Thakera, Joseph Adelsbergerd,
Adam Rupertd, Michael Baselerd, Yutaka Tagayae, Gregg Robya, Catherine Rehma, Dean Follmannb,
and H. Clifford Lanea
aClinical and Molecular Retrovirology Section, Laboratory of Immunoregulation andbBiostatistics Research Branch, National Institute of Allergy and
Infectious Diseases, National Institutes of Health, Bethesda, MD 20892;cBiostatistics Research Branch anddAIDS Monitoring Laboratories, Science
Applications International Corporation, Frederick, MD 21702; andeMetabolism Branch, Center for Cancer Research, National Cancer Institute,
National Institutes of Health, Bethesda, MD 20892
Communicated by Anthony S. Fauci, National Institutes of Health, Bethesda, MD, October 27, 2008 (received for review July 7, 2008)
HIV infection is characterized by a brisk immune activation that
plays an important role in the CD4 depletion and immune dysfunc-
tion of patients with AIDS. The mechanism underlying this activa-
tion is poorly understood. In the current study, we tested the
hypothesis that this activation is the net product of two distinct
pathways: the inflammatory response to HIV infection and the
homeostatic response to CD4 T cell depletion. Using ex vivo BrdU
incorporation of PBMCs from 284 patients with different stages of
HIV infection, we found that CD4 proliferation was better pre-
0.375, P < 0.001) than by either parameter alone (CD4 T cell counts,
R2? 0.202, P < 0.001; HIV viremia, R2? 0.302, P < 0.001).
Interestingly, CD8 T cell proliferation could be predicted by HIV
RNA levels alone (R2? 0.334, P < 0.001) and this predictive value
increased only slightly (R2? 0.346, P < 0.001) when CD4 T cell
depletion was taken into account. Consistent with the hypothesis
that CD4 T cell proliferation is driven by IL-7 as a homeostatic
response to CD4 T cell depletion, levels of phosphorylated STAT-5
were found to be elevated in naive subsets of CD4 and CD8 T cells
from patients with HIV infection and in the central memory subset
of CD4 T cells. Taken together these data demonstrate that at least
two different pathways lead to immune activation of T cells in
patients with HIV infection and these pathways differentially
influence CD4 and CD8 T cell subsets.
CD4 T cell homeostasis ? IL-7 ? STAT-5
immune system. While a direct infection of CD4 T cells by HIV
partially explains the CD4 T cell depletion, it is clear that the
overall disruption of immune function in patients with HIV
infection is the sum of multiple factors (1). Immune activation is
a major contributor to the pathogenesis of HIV disease and is
manifested in many ways varying from increased T cell prolif-
eration, as reflected in an increased number of cycling cells as
measured by DNA incorporation of BrdU or intranuclear ex-
pression of Ki67, to increased expression of surface activation
markers such as HLA-DR and CD38 (2–4). The importance of
immune activation in patients with HIV infection is reflected in
the observation that increased expression of CD38 on CD4 and
CD8 T cells has been found to be a better correlate of clinical
disease progression than CD4 T cell counts or HIV-RNA levels
Despite the presence of immunodeficiency, virtually all cel-
lular components of the immune system, B cells, NK cells, T cells
and macrophages show evidence of immune activation (5, 7–9).
Elevated CD4 and CD8 T cell proliferation can be observed in
vitro and in vivo through examination of Ki67, measurement of
DNA content, or labeling with BrdU (10–15). Increased levels
IV infection is characterized by chronic immune activation
and CD4 T cell depletion leading to dysfunction of the
of proinflammatory cytokines including forms of IFN-?, TNF-?
and IL-6 are also present in patients with HIV infection (16–18).
Whether this is directly or indirectly part of the host response to
HIV is unclear. In addition, in HIV infection and other lym-
phopenic conditions, depletion of CD4 T cells triggers homeo-
static responses that can result in increased circulating levels of
IL-7 (19–22). IL-7 is a member of the type I cytokine family and
signals through a heterodimer receptor composed of the IL-7R?
chain and the common cytokine signaling ?-chain present in
receptors for IL-2, IL-4, IL-7, IL-9, IL-15 and IL-21 (23). The
IL-7R complex signals through the Janus kinases, JAK1 and
JAK3, leading to phosphorylation of the signal transducer and
activator of transcription-5 (STAT-5). Phosphorylated dimers of
STAT-5 translocate to the nucleus and induce gene transcription
and cell cycle progression (24, 25).
HIV infection is that while both CD4 and CD8 T cells are
activated, one sees depletion of the CD4 T cell pool and
expansion of the CD8 T cell pool. Although the direct cytopathic
the low number of cells actively infected at any given point in
time makes this an unsatisfactory explanation of the huge
dichotomy seen between these two subsets. One alternative
hypothesis is that different pathways of activation are triggered
studies have shown that proliferation of CD4 and CD8 T cell
subsets correlate with plasma levels of HIV RNA, however, the
role of the T cell homeostatic response to CD4 T cell depletion
has not been fully analyzed. We hypothesized that CD4 T cell
depletion leads to a homeostatic response occurring in an
inflammatory environment generated and maintained by the
virus. In such a setting, CD4 T cells could be induced to
proliferate by both forces with the net result being activation
induced cell death. In the present study, we sought to determine
the relative contributions of viral load and its associated inflam-
matory environment and CD4 T cell depletion and its associated
Author contributions: M.C., and H.C.L. designed research; M.C., V.T., G.R., and C.R. per-
formed research; M.D.M., J.A., A.R., M.B., Y.T., and D.F. contributed new reagents/analytic
tools; M.C., M.D.M., Z.H., and S.S. analyzed data; and M.C., M.D.M., D.F., and H.C.L. wrote
The authors declare no conflict of interest.
Freely available online through the PNAS open access option.
1To whom correspondence should be addressed at: Clinical and Molecular Retrovirology
Section, Laboratory of Immunoregulation, National Institute of Allergy and Infectious
Diseaases, National Institutes of Health, Bldg. 10 Room 7N246, 9000 Rockville Pike,
Bethesda, MD 20892-1360. E-mail: firstname.lastname@example.org.
This article contains supporting information online at www.pnas.org/cgi/content/full/
© 2008 by The National Academy of Sciences of the USA
December 16, 2008 ?
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homeostatic response on CD4 and CD8 T cell activation in
patients with HIV infection by examining the relationships
between CD4 counts, HIV RNA levels and BrdU incorporation.
By virtue of being a selective marker of cells in S-phase and thus
a measurement of cells actively proliferating, examination of
BrdU incorporation allows a detailed analysis of that element of
immune activation reflected in increased T cell proliferation.
Study Cohort. The study cohort consisted of 284 patients with
HIV infection seen during the period of February 1998 to
February 2007. Their CD4 T cell counts ranged from 11 to 1938
cells per microliter, and their HIV RNA levels from ?50 to
860,608 copies per milliliter. At the time of enrollment, the
CD8 T cell count was 839 cells per microliter and the median
HIV RNA level was 56 copies per milliliter. Patients were
monitored for a median of 7.5 visits. The median CD4 T cell and
CD8 T cell counts for the cohort of normal volunteers (n ? 373)
were 846 cells per microliter and 392 cells per microliter
respectively. The normal volunteers were monitored for a me-
dian of 2 visits [The datasets used for each analysis are depicted
in supporting information (SI) Table S1].
CD4 Depletion Drives CD4 T Cell Proliferation in both Normal Volun-
teers and Patients with HIV Infection. To examine the separate
effects of CD4 depletion and HIV viral load on T cell prolifer-
ation, we measured spontaneous ex vivo incorporation of BrdU
in the CD4 and CD8 T cells from the whole blood of patients
with HIV infection; characterized as having high (?800 cells per
microliter) or low (?300 cells per microliter) levels of CD4 T
cells and high (?10,000 copies per milliliter) or low (?50 copies
per milliliter) levels of HIV RNA (Fig. 1A). Similar determina-
tions were performed on cells from normal volunteers with high
(?800 cells per microliter) or low (?500 cells per microliter)
CD4 T cell counts (Fig. 1B).
Both normal volunteers and patients with HIV infection and
HIV RNA levels ?50 copies per milliliter showed an increase in
CD4 proliferation when those with higher CD4 counts were
compared with those with lower CD4 counts [0.053 vs. 0.075%
(P ? 0.001)] for normal volunteers and [0.085 vs. 0.203% (P ?
0.001)] for patients (Fig. 1 A and B). The absolute CD4 T cells
counts had little absolute effect on CD8 proliferation of the 2
HIV groups with ?50 copies per milliliter [0.075 vs. 0.045% (P ?
0.001)] and in normal volunteers [0.033 vs. 0.035% (P ? 0.05)].
In both HIV infected groups with HIV-RNA levels ?50
copies per milliliter and normal volunteers, CD4 T cell prolif-
eration was higher than CD8 T cell proliferation (P ? 0.001 in
the HIV groups and P ? 0.006 and P ? 0.001 in normal
volunteers, respectively Fig. 1 A and B). This effect was also
observed in the group of patients with low CD4 counts and high
viral loads (P ? 0.004). In contrast, CD8 T cell proliferation
trended higher than CD4 T cell proliferation in the HIV patients
with high CD4 T cell counts and high viral load.
The relationship between CD4 T cell depletion and CD4 and
CD8 T cell proliferation was further analyzed in those patients
with viral loads ?50 copies per milliliter and in normal volun-
teers (Fig. 2A). Lower CD4 T cell counts were associated with
increased rates of CD4 T cell proliferation in both HIV infected
and healthy volunteers. The slopes describing these relationships
indicated higher rates of CD4 T cell proliferation for patients
with HIV infection. Similar rates of CD4 T cell proliferation at
very high CD4 T cell counts (1,500 cells per microliter) were
observed for HIV infected patients and controls. Of note, there
was no intercept for the CD8 lines. Thus, a persistent force
independent of CD4 counts, probably ‘‘ongoing’’ viral replica-
tion leads to higher rates of CD8 proliferation in all patients with
HIV infection. This includes those patients with ‘‘undetectable’’
HIV RNA levels.
HIV Viral Load Drives both CD4 and CD8 Proliferation in Patients with
HIV Infection. A comparison of rates of T cell proliferation in
patients with HIV infection separated into cohorts based upon
viral load revealed higher rates of both CD4 and CD8 prolifer-
ation as a function of increased viral load (Fig. 1A). For CD4
proliferation, the cohort of patients with low (?300 cells per
microliter) CD4 counts demonstrated approximately a 3-fold
increase in rates of BrdU incorporation when patients with HIV
depletion in HIV infected individuals and normal volunteers, while CD8 T cell
proliferation is driven by the viral load. (A) Ex vivo BrdU labeling of PBMCs in
a cohort of 284 HIV positive individuals. T cells were analyzed by flow cytom-
etry gating in CD3?CD4?and CD3?CD8?T cells. The groups of patients were
divided based upon low (LCD4: ?300 cells per microliter) and high (HCD4:
?800 cells per microliter) CD4 T cells with high (HVL: ?10,000 copies per
milliliter) and with low (LVL: ?50 copies per milliliter) viral loads. (B) Ex vivo
BrdU labeling of PBMCs from normal volunteers (n ? 373) as above. The
normal volunteers were divided into individuals with low (LCD4: ?500 cells
per microliter) and high (HCD4: ?800 cells per microliter) CD4 T cell counts.
Values for CD4 T cell counts and viral load are expressed as Mean (SD) across
T cell proliferation.
www.pnas.org?cgi?doi?10.1073?pnas.0810032105Catalfamo et al.
RNA levels of ?50 copies per milliliter were compared with
those with HIV RNA levels ?10,000 copies per milliliter (0.203
on CD4 proliferation in patients with high (?800 cells per
microliter) CD4 T cell counts with only a 1.7-fold increase in
BrdU incorporation observed in this subset of patients as a
consequence of higher viral loads (0.085 vs. 0.148%, P ? 0.001).
For CD8 proliferation, both cohorts of patients with high
(?10,000 copies per milliliter) viral loads had rates of BrdU
incorporation that were ?5- and 6-fold higher than their respec-
tive CD4 cohorts with low (?50 copies per milliliter) viral loads
(0.269 vs. 0.045%, P ? 0.001 and 0.396 vs. 0.075%, P ? 0.001; for
high CD4 and low CD4 T cell count groups respectively).
Viral Load and CD4, CD8 T Cell Proliferation. Taken together these
data indicate that CD4 proliferation is the net result of immune
activation driven by viral load and homeostatic forces while CD8
proliferation is mainly driven by viral load. The critical role of
CD4 T cell depletion in CD4 T cell proliferation is suggested by
those patients in whom CD4 T cell counts are ?800 cell/?l yet
whose viral loads are ?10,000 copies per milliliter. In these
patients, CD8 proliferation numerically outpaced CD4 prolifer-
ation (0.269% vs. 0.148%, P ? 0.094, Fig. 1A). To better define
the above relationships, we performed a multivariate analysis
with mixed-effects linear models, using CD4 count and viral load
as covariates and CD4 and CD8 proliferation as reflected in
BrdU incorporation as the readouts (Table 1 and Table S1).
Together CD4 count and viral load could account for 37.5% of
the variance in CD4 T cell proliferation and 34.6% of the
variance in CD8 T cell proliferation. For CD4 proliferation the
predictive value of viral load alone was 30.2% and increased to
37.5% with the addition of CD4 count. In contrast, for CD8
proliferation the predictive value of viral load alone was 33.4%
and increased very little (to 34.6%) with the addition of CD4
count to the model. These relationships are depicted graphically
in Fig. 2B in which one can observe that CD4 proliferation is
of high CD4 count and high viral load.
In Vivo Contribution of Cytokines to T Cell Proliferation. Having
established the fact that CD4 proliferation is driven by both CD4
depletion (homeostasis) and viral load while CD8 proliferation
is mainly driven by viral load we sought to determine the role,
if any, played by cytokines known to be associated with T cell
homeostasis. Levels of the ?-common cytokines IL-2, IL-7 and
IL-15 were determined for a computer generated set of 102
S1) to ensure a even split among the 4 groups of patients. As
demonstrated in Fig. 3A, whereas levels of IL-7 were highest in
the two groups of patient with the lowest CD4 counts, levels of
IL-15 were similar among all 4 groups. For IL-2, median serum
levels were similar between the groups (HCD4-HVL: 39.1 pg/ml;
LCD4-LVL: 38.4 pg/ml; LCD4-HVL: 38.4 pg/ml; HCD4-LVL:
38.4 pg/ml). Using an univariate linear regression analysis, the
relationships between IL-7 levels and CD4 or CD8 T cell counts,
showed a strong inverse correlation between CD4 counts and
serum IL-7 levels (R2? 0.36; P ? 0.001); and no correlation
between CD8 counts and IL-7 levels (R2? 0.02; P ? 0.14). Since
viral load has been associated with higher serum levels of IL-7
HIV RNA levels ?50 copies per milliliter (Table S2). In this
and CD4 T cell depletion. (A) The relationship between CD4 T cell count and
CD4 (CD4 BrdU?T cells) or CD8 (CD8 BrdU?T cells) proliferation was studied
and normal volunteers. Linear regressions of T cell proliferation vs. CD4 T cell
count are depicted in the solid lines with the 95% confidence interval bands
for the normal volunteers are depicted in blue. The P values indicate the
differences between the slopes (ANCOVA type analysis). (B) The relationships
Table 1. Mutivariate analysis of CD4 and CD8 T cell proliferation with the covariates CD4 T cell count and viral load
Coef of CD4
(cell per microliter)?1
Coeficient of VL
(95% CI), (copies per
P (univariated vs.
Catalfamo et al.
December 16, 2008 ?
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subset, the multivariate analysis indicated that CD4 T cell counts
(R2? 0.42, P ? 0.001) were a better predictor of IL-7 serum
levels than the viral loads (R2? 0.22, P ? 0.012). To further
examine the relationship between viral loads, CD4 counts and
serum IL-7 levels, an ANCOVA analysis was carried out using
the 102 samples selected for cytokine measurements (Table S1).
In this analysis, viral load was treated as a binary variable
(?10,000 or ?50 copies per milliliter) while adjusting for the
CD4 counts. This analysis, depicted in Fig. 3B, indicated that at
lower CD4 counts (?300 cells per microliter), patients with high
viral loads tended to have higher serum levels of IL7 than
patients with low viral loads (P ? 0.05). No such association was
seen for patients with higher CD4 counts (?800 cells per
The relative contributions of the covariates CD4 count, IL-7
and HIV-RNA copy number to CD4 and CD8 T cell prolifer-
?50 copies per milliliter (Table S3). In this cohort, CD4 T cell
proliferation was most strongly correlated to CD4 counts (R2?
in addition to IL-7 may be driving homeostasis. As was expected
from the previous results, CD8 T cell proliferation correlated
most strongly with the HIV-RNA levels (R2? 0.269).
To determine whether or not the proinflammatory cytokines
that have been associated with ongoing HIV replication are
contributing to CD8 proliferation, analyses for IFN-?, TNF-?,
IL-1?, IL-2, IL-4, IL-5, IL-8, IL-10, IL-12p70, and IL-13 were
performed on the same 102 samples selected above (Table S1).
Although correlations were noted between IFN-? levels and
milliliter (R2? 0.22; P ? 0.012), IFN-? did not add predictive
value to CD8 T cell proliferation beyond that seen for viral load
alone (data not shown), suggesting that factors other than those
studied here may be leading to CD8 proliferation in the setting
of HIV infection (Table S2).
Increased STAT-5 Phosphorylation in T Cells of HIV Infected Individ-
uals. The above data suggest that IL-7, associated with low CD4
T cell counts, may be playing a role in the homeostatic induction
of CD4 proliferation and may play some slight role in CD8
proliferation. To further investigate this relationship we exam-
ined peripheral blood T cells for evidence of recent IL-7
signaling. Early events after engagement of the IL-7 receptor by
IL-7 include activation of Janus family kinases JAK1 and JAK3
and phosphorylation of cytoplasmic STAT-5 to generate
in vivo one would expect freshly isolated T cells to express
increased levels of pSTAT5 before cell cycle entry (27, 28).
Accordingly, peripheral blood mononuclear cells from 34 HIV
infected individuals and 18 normal volunteers were examined for
the presence or absence of intracellular pSTAT5 by flow cytom-
intensity for pSTAT5 staining were noted between CD8 T cells
from HIV infected individuals and healthy controls; increased
levels of pSTAT5 were noted in the CD4 cells from HIV infected
individuals when compared with controls (P ? 0.019). The
hundred two serum samples from the 4 groups of HIV positive patients were
tested by ELISA for IL-7 (red symbols) and IL-15 (blue symbols). Values for CD4
T cell counts and viral load are expressed as means (SD). Median values for the
IL-7 levels are noted inside the plot. (B) Linear regression of IL-7 vs. CD4 T cell
counts in patients with viral loads ?10,000 (solid red line) or ?50 (solid blue
line) copies per milliliter. Dotted lines represent the 95% confidence interval
Effect of CD4 T cell counts and viral load on IL-7 serum levels. (A) One
normal volunteers. (A)Whole blood was stained by flow cytometry for
pSTAT-5 in normal volunteers and HIV positive individuals. (B) Results from
an individual subject (34 HIV positive and 18 normal individuals). The mean
mean (SD) CD8 counts for the patients and controls were 870 (518) and 376
(237); and the mean(SD) HIV RNA level for the patients was 35,861 (90, 228).
Phosphorylated STAT-5 expression in HIV positive individuals and
www.pnas.org?cgi?doi?10.1073?pnas.0810032105 Catalfamo et al.
presence of pSTAT-5 was noted to be restricted to the Ki67
negative population of cells. This lack of pSTAT-5 in the Ki67?
cell population reflects the early, transient nature of pSTAT-5
expression. These data support the hypothesis that IL-7 is involved
and suggest that common gamma chain using cytokines are not a
major factor in the overall proliferation of CD8 T cells.
Of note, phosphorylated levels of STAT-5 were higher in CD4
T cells than CD8 T cells (P ? 0.001) in both HIV positive and
healthy volunteers. We did not find any correlation between the
MFI of pSTAT-5 staining and serum IL-7 levels. This lack of
correlation may be due to the small sample size studied or the
presence of other factors. Thus, the precise relationship between
these 2 measurements is difficult to ascertain. To determine
whether the increased pSTAT-5 phosphorylation observed in
CD4 T cells was driven by a particular subset of CD4 T cells we
measured the MFI of pSTAT-5 in naive (N), central memory
(CM), effector memory (EM), and terminal effector memory T
cells (TEM) based on the expression of CD45RA and CD62L.
Naive and central memory CD4 T cells and naive CD8 T cells,
showed significantly increased levels of pSTAT-5, compared
with normal volunteers (Fig. 5).
Human HIV infection disrupts the immune system through
generalized immune activation and CD4 T cell depletion. In the
present study, we demonstrate that immune activation as re-
flected in T cell proliferation is driven by both homeostatic and
CD4 T cell proliferation is driven by the homeostatic response to
CD4 T cell depletion and by viremia, whereas CD8 T cell prolif-
able to demonstrate for the CD4 T cell pool that the homeostatic
proliferation induced by CD4 T cell depletion is accelerated by the
inflammatory environment generated by the virus.
T cell homeostasis is an important mechanism to assure
survival and maintenance of the T cell repertoire through life.
Key regulators of this process are the gamma-common cytokines
IL-2, IL-7 and IL-15 (21, 22). Increased serum levels of IL-7 have
been described in patients with HIV infection and in other
correlations observed between CD4 depletion, increased T cell
proliferation and IL-7 levels (levels of IL-2 and IL-15 were not
significantly different between the groups) suggest that homeo-
static forces represent an important factor in the immune
activation seen in patients with HIV infection. This statement is
supported by the increased levels of pSTAT-5 seen in naive and
central memory CD4 T cells and in naive CD8 T cells. The
elevated levels of pSTAT-5 observed in the naive CD4 and CD8
T cell compartments compared with the levels in cells from
levels of IL-7 observed in HIV infection (ref. 19 and Fig. 3B).
The lack of STAT-5 phosphorylation in the other T cell subsets
is consistent with the hypothesis that their proliferation is
primarily driven by different forces. These forces are likely
directly induced by the virus and may include things such as TcR
specific expansions and/or bystander inflammatory cytokines.
Given that the differentiation pathway to effector T cells leads
to a loss of CD127 and a refractoriness of CD8 effector T cells
to IL-7 signaling (30), these results are not surprising. pSTAT-5
was observed predominantly in Ki67-negaive cells, which sup-
ports the notion that phosphorylation of STAT-5 is an early and
transient marker of cell-cycle progression.
HIV viremia has been associated with a clear effect on the
activation and proliferation of both CD4 and CD8 T cells (14).
In the present study, viral load alone predicted 30.2% and 33.4%
of CD4 and CD8 T cell proliferation respectively. These are
substantially higher predictive values than the 10% that has been
associated with the ability of viral load to predict CD4 decline
(31). The downstream pathways leading to this proliferation
remain unclear but are likely a combination of both specific TcR
engagement and non specific bystander activation via inflam-
matory cytokines such as plasmacytoid DC secreted type I IFN,
IFN-? and TNF-?, and/or microbial translocation of LPS (7, 17,
32). HIV viremia perpetuates an inflammatory milieu (33),
likely causing a bystander effect mediated by cytokines, as has
been described in vitro with human CD4 T cells (33, 34). The
effect of inflammation on the proliferation of CD4 and CD8 T
cells is quickly reduced when viremia is suppressed by HAART.
Unfortunately, none of the cytokines tested in this study showed
a relationship to CD8 T cell proliferation, suggesting that other
cytokines or mechanisms are contributing to this aspect of
These results suggesting two forces driving CD4 proliferation
are consistent with in vivo labeling experiments of HIV infected
individuals with BrdU, which showed a biphasic kinetics of CD4
and CD8 T cells, including ‘‘rapidly’’ and ‘‘slowly’’ proliferating
populations (13, 35). In this situation the CD4 T cells driven by
homeostatic forces into the slowly proliferating pool may be
more easily recruited into the ‘‘rapidly’’ proliferating pool where
they undergo additional proliferation by bystander mechanisms
and ultimately die. After initiation of HAART, the ‘‘rapidly’’
proliferating (and dying) CD4 T cell population, is selectively
HIV positive individuals and normal volunteers. (A) Whole blood was stained
by flow cytometry for pSTAT-5 in normal volunteers and HIV positive individ-
uals, using CD45RA and CD62L as markers of naive, central memory, effector
individual subject (16 HIV positive and 16 normal individuals).
Catalfamo et al.
December 16, 2008 ?
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no. 50 ?
and immediately reduced while there is little effect on the Download full-text
could help to explain why one sees a rapid increased followed by
a slow, steady increase in the size of the CD4 T cell pool after
the initiation of HAART.
The results from the normal volunteer cohort, revealed a clear
inverse correlation between CD4 T cell proliferation and CD4 T
cell counts with less of an effect of CD4 T cell counts on the
proliferation of the CD8 T cells (Figs. 1B and 2A). These data
illustrate the increased importance of homeostasis in mainte-
nance of the CD4 T cell pool and are consistent with studies in
SIV infected and uninfected sooty mangabeys (36). Levels of
CD4 proliferation in HIV positive patients approached that of
HIV negative controls as the CD4 T cell counts increased;
confirming the more potent effects of homeostatic than virologic
forces on the CD4 T cell pool. In contrast, CD8 T cell prolif-
eration was elevated in all patients with HIV infection, demon-
strating a strong correlation with viremia even in HIV infected
pathways leading to CD8 proliferation remain unclear. CD8 T cells
with non-HIV specificities have also been shown to express an
bystander activation in the setting of HIV infection.
These data contribute to our understanding of the effects that
HIV infection has on the CD4 and CD8 T cell pools. In the case
of CD4 T cells one has virus-specific immune responses and
inflammatory forces in the presence of homeostatic forces with
the net result being activation and slow CD4 T cell depletion. In
the case of CD8 T cells, one has virus-specific immune responses
and inflammatory forces with the net result being CD8 T cell
expansion. Further study of the precise mechanisms involved
may help to identify new targets for therapeutic intervention.
Patient Selection. From February 1998 to February 2007, patients and controls
studied in National Institute of Allergy and Infectious Diseases/Critical Care
Medicine Department intramural IRB approved HIV clinical research studies
had routine measurement of T cell turnover/immune activation including cell
surface staining for HLA-DR, CD38, CD25, nuclear antigen Ki67 and sponta-
neous incorporation of BrdU. During this time, a total of 341 patients and 373
S1 summarizes the datasets for the analysis and the description of the ana-
lytical methods). The majority of the patients studied had chronic HIV infec-
tion (?5% with acute infection) and ?75% received a variety of standard of
care antiretroviral regimens over the 10-year period of the study. By pooling
together a set of treated and untreated patients and thus creating a hetero-
geneous group for study we were able to highlight the independence of the
of STAT-5 phosphorylation.
Ex Vivo BrdU Labeling. Whole blood was labeled as described in ref. 14.
Cytokine Measurements. Serum samples from the HIV positive groups were
tested by ELISA for IL-2 (Pierce, Rockford, IL), IL-7 and IL-15 (R&D Systems) and
IFN-alpha (Amersham Biosciences). A 10 multiplex kit (Meso Scale Discovery)
Flow Cytometry. Detection of phosphorylated STAT-5 (pSTAT-5) was per-
formed by flow cytometry as described in ref. 39. The following mAbs were
used anti-CD3, CD4, Ki67, CD45RA, CD62L and pSTAT-5 (BD Biosciences). The
FlowJo software (Ashland).
Supporting Information. The datasets used for analysis and statistical methods
are described in SI Text.
ACKNOWLEDGMENTS. We thank the patients of the National Institute of
Allergy and Infectious Diseases HIV-Clinic for their participation in this study,
Anthony Fauci for his guidance and support. This work was supported by the
Intramural Research Program of the National Institute of Allergy and Infec-
tious Diseases, National Institutes of Health; National Cancer Institute, Na-
tional Institutes of Health Contract N01-CO-12400 (to Science Applications
infection: How CD4? T cells go out of stock. Nat Immunol 1:285–289.
2. Ho HN, et al. (1993) Circulating HIV-specific CD8? cytotoxic T cells express CD38 and
HLA-DR antigens. J Immunol 150:3070–3079.
3. Kestens LG, et al. (1994) Selective increase of activation antigens HLA-DR and CD38 on
CD4? CD45RO? T lymphocytes during HIV-1 infection. Clin Exp Immunol 95:436–441.
4. Mahalingam M, et al. (1993) T cell activation and disease severity in HIV infection. Clin
Exp Immunol 93:337–343.
5. Giorgi JV, et al. (1999) Shorter survival in advanced human immunodeficiency virus
type 1 infection is more closely associated with T lymphocyte activation than with
plasma virus burden or virus chemokine coreceptor usage. J Infect Dis 179:859–870.
6. Hazenberg MD, et al. (2003) Persistent immune activation in HIV-1 infection is associ-
ated with progression to AIDS. AIDS 17:1881–1888.
7. Brenchley JM, et al. (2006) Microbial translocation is a cause of systemic immune
activation in chronic HIV infection. Nat Med 12:1365–1371.
8. Lane HC, et al. (1983) Abnormalities of B-cell activation and immunoregulation in
patients with the acquired immunodeficiency syndrome. N Engl J Med 309:453–458.
9. Fauci AS, Mavilio D, Kottilil S (2005) NK cells in HIV infection: Paradigm for protection
or targets for ambush. Nat Rev Immunol 5:835–843.
10. Sachsenberg N, et al. (1998) Turnover of CD4? and CD8? T lymphocytes in HIV-1
infection as measured by Ki-67 antigen. J Exp Med 187:1295–1303.
11. Zhang ZQ, et al. (1998) Kinetics of CD4? T cell repopulation of lymphoid tissues after
treatment of HIV-1 infection. Proc Natl Acad Sci USA 95:1154–1159.
12. Ho DD, et al. (1995) Rapid turnover of plasma virions and CD4 lymphocytes in HIV-1
infection. Nature 373:123–126.
13. Kovacs JA, et al. (2001) Identification of dynamically distinct subpopulations of T
lymphocytes that are differentially affected by HIV. J Exp Med 194:1731–1741.
14. Lempicki RA, et al. (2000) Impact of HIV-1 infection and highly active antiretroviral
Natl Acad Sci USA 97:13778–13783.
15. Sieg SF, et al. (2005) Peripheral S-phase T cells in HIV disease have a central memory
phenotype and rarely have evidence of recent T cell receptor engagement. J Infect Dis
16. Smed-Sorensen A, et al. (2005) Differential susceptibility to human immunodeficiency
17. Herbeuval JP, et al. (2006) Differential expression of IFN-alpha and TRAIL/DR5 in
lymphoid tissue of progressor versus nonprogressor HIV-1-infected patients. Proc Natl
Acad Sci USA 103:7000–7005.
18. Tilton JC, et al. (2006) Diminished production of monocyte proinflammatory cytokines
19. Napolitano LA, et al. (2001) Increased production of IL-7 accompanies HIV-1-mediated
T-cell depletion: Implications for T-cell homeostasis. Nature medicine 7:73–79.
20. Fry TJ, et al. (2001) A potential role for interleukin-7 in T-cell homeostasis. Blood
maintenance. J Immunol 174:6571–6576.
22. Mackall CL, Hakim FT, Gress RE (1997) Restoration of T-cell homeostasis after T-cell
depletion. Semin Immunol 9:339–346.
23. Puel A, Ziegler SF, Buckley RH, Leonard WJ (1998) Defective IL7R expression in T(?)
B(?)NK(?) severe combined immunodeficiency. Nat Genet 20:394–397.
24. Mazzucchelli R, Durum SK (2007) Interleukin-7 receptor expression: Intelligent design.
Nat Rev Immunol 7:144–154.
25. Gadina M, et al. (2001) Signaling by type I and II cytokine receptors: Ten years after.
Current opinion in immunology 13:363–373.
26. O’Shea JJ, Gadina M, Schreiber RD (2002) Cytokine signaling in 2002: New surprises in
the Jak/Stat pathway. Cell 109:S121–S131.
27. Yu CL, Jin YJ, Burakoff SJ (2000) Cytosolic tyrosine dephosphorylation of STAT5.
Potential role of SHP-2 in STAT5 regulation. J Biol Chem 275:599–604.
28. Seki Y, et al. (2007) IL-7/STAT5 cytokine signaling pathway is essential but insufficient
for maintenance of naive CD4 T cell survival in peripheral lymphoid organs. J Immunol
29. Malaspina A, et al. (2007) Idiopathic CD4? T lymphocytopenia is associated with
increases in immature/transitional B cells and serum levels of IL-7. Blood 109:2086–
30. Boyman O, Purton JF, Surh CD, Sprent J (2007) Cytokines and T-cell homeostasis. Curr
Opin Immunol 19:320–326.
decline in untreated HIV infection. JAMA 296:1498–1506.
are associated with HIV infection. J Immunol 151:5031–5040.
33. Boasso A, Shearer GM (2008) Chronic innate immune activation as a cause of HIV-1
immunopathogenesis. Clin Immunol 126:235–242.
34. Geginat J, Sallusto F, Lanzavecchia A (2001) Cytokine-driven proliferation and differ-
entiation of human naive, central memory, and effector memory CD4(?) T cells. J Exp
35. Hellerstein, et al. (2003) Subpopulations of long-lived and short-lived T cells in ad-
vanced HIV-1 infection. J Clin Invest 112:956–966.
36. Kaur A, et al. (2008) Dynamics of T- and B-lymphocyte turnover in a natural host of
simian immunodeficiency virus. J Virol 82:1084–1093.
37. Doisne JM, et al. (2004) CD8? T cells specific for EBV, cytomegalovirus, and influenza
virus are activated during primary HIV infection. J Immunol 173:2410–2418.
38. Tough DF, Borrow P, Sprent J (1996) Induction of bystander T cell proliferation by
viruses and type I interferon in vivo. Science 272:1947–1950.
using polychromatic flow cytometry. Nat Biotechnol 20:155–162.
www.pnas.org?cgi?doi?10.1073?pnas.0810032105Catalfamo et al.