A Low T Regulatory Cell Response May Contribute to
Both Viral Control and Generalized Immune Activation in
Peter W. Hunt1*, Alan L. Landay2, Elizabeth Sinclair1, Jeffrey A. Martinson2, Hiroyu Hatano1, Brinda
Emu1, Philip J. Norris1,3, Michael P. Busch1,3, Jeffrey N. Martin1, Cicely Brooks2, Joseph M. McCune1,
Steven G. Deeks1
1Departments of Medicine and Laboratory Medicine, University of California San Francisco, San Francisco, California, United States of America, 2Department of
Immunology/Microbiology, Rush University Medical Center, Chicago, Illinois, United States of America, 3Blood Systems Research Institute, San Francisco, California,
United States of America
HIV-infected individuals maintaining undetectable viremia in the absence of therapy (HIV controllers) often maintain high
HIV-specific T cell responses, which has spurred the development of vaccines eliciting HIV-specific T cell responses.
However, controllers also often have abnormally high T cell activation levels, potentially contributing to T cell dysfunction,
CD4+ T cell depletion, and non-AIDS morbidity. We hypothesized that a weak T regulatory cell (Treg) response might
contribute to the control of viral replication in HIV controllers, but might also contribute to generalized immune activation,
contributing to CD4+ T cell loss. To address these hypotheses, we measured frequencies of activated (CD38+ HLA-DR+),
regulatory (CD4+CD25+CD127dim), HIV-specific, and CMV-specific T cells among HIV controllers and 3 control populations:
HIV-infected individuals with treatment-mediated viral suppression (ART-suppressed), untreated HIV-infected ‘‘non-
controllers’’ with high levels of viremia, and HIV-uninfected individuals. Despite abnormally high T cell activation levels,
controllers had lower Treg frequencies than HIV-uninfected controls (P=0.014). Supporting the propensity for an unusually
low Treg response to viral infection in HIV controllers, we observed unusually high CMV-specific CD4+ T cell frequencies and
a strong correlation between HIV-specific CD4+ T cell responses and generalized CD8+ T cell activation levels in HIV
controllers (P#0.001). These data support a model in which low frequencies of Tregs in HIV controllers may contribute to an
effective adaptive immune response, but may also contribute to generalized immune activation, potentially contributing to
Citation: Hunt PW, Landay AL, Sinclair E, Martinson JA, Hatano H, et al. (2011) A Low T Regulatory Cell Response May Contribute to Both Viral Control and
Generalized Immune Activation in HIV Controllers. PLoS ONE 6(1): e15924. doi:10.1371/journal.pone.0015924
Editor: Mario Ostrowski, University of Toronto, Canada
Received July 28, 2010; Accepted November 30, 2010; Published January 31, 2011
Copyright: ? 2011 Hunt 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: This work was supported in part by the UCSF/Gladstone Center for AIDS Research (P30 AI27763, P30 MH59037); NIAID (AI065244, AI055273, AI44595,
AI067854, AI076981, and AI-76174); the Center for AIDS Prevention Studies (P30 MH62246); the UCSF Clinical and Translational Science Institute (UL1 RR024131-
01); the CFAR Network of Integrated Clinical Sciences (5R24AI067039); the Ragon Institute of MGH, MIT, and Harvard; and American Foundation for AIDS Research
(106710-40-RGRL). JMM is a recipient of a grant (DPI OD00329) from the NIH Director’s Pioneer Award Program, part of the NIH Roadmap for Medical Research.
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
The HIV vaccine field has returned ‘‘back to basics’’ after a T
cell-mediated immunity vaccine recently failed to prevent HIV
infection and actually increased the risk of infection in important
subgroups of individuals . Part of this process is a re-
examination of the mechanisms by which some HIV-infected
individuals spontaneously control viral replication in the absence
of antiretroviral therapy. These HIV controllers represent fewer
than 1% of chronically HIV-infected individuals and maintain
clinically undetectable plasma HIV RNA levels (operationally
defined as ,75 copies/ml) in the absence of antiretroviral
medications [2,3,4,5]. Several functional immunologic and host
genetics studies suggest that high levels of HIV-specific CD4+ and
CD8+ T cells with preserved function are likely to play an
important role in the suppression of viral replication in most of
these individuals [6,7,8,9,10,11,12,13,14,15,16,17,18,19], obser-
vations which have spurred the development of T cell immunity
vaccines for HIV. However, the mechanisms of viral control in
these individuals are likely to be heterogeneous as many HIV
controllers lack a protective HLA type, have very low frequencies
of HIV-specific T cells, or maintain control of viral replication
even after documented escape from HLA-restricted epitopes
It is important to recognize this heterogeneity as some
mechanisms of viral control may prevent both initial infection
and clinical progression better than others. For example, high T
cell activation and low regulatory T cell (Treg) responses in highly
exposed HIV-uninfected individuals have been consistently
associated with an increased risk of subsequent HIV infection
[23,24,25,26,27]. Higher T cell activation has also been
independently associated with more rapid CD4+ T cell decline
and clinical progression to AIDS in untreated HIV-infected
individuals [28,29,30,31,32,33]. This potentially harmful effect of
PLoS ONE | www.plosone.org1 January 2011 | Volume 6 | Issue 1 | e15924
activation has even been observed among controllers .
Persistent immune activation in HIV controllers may also
contribute to accelerated atherosclerosis and other non-AIDS
morbidities linked to inflammation . Understanding why some
mechanisms of viral control are associated with negative
inflammatory consequences is therefore an important issue for
HIV vaccine development.
We hypothesized that an unusually low Treg response to viral
infection might allow some HIV controllers to maintain strong
antiviral immune responses at the cost of at the cost of abnormally
high generalized immune activation, potentially contributing to
CD4+ T cell decline even in the absence of clinically detectable
viremia. To address these hypotheses, we measured frequencies
of activated (CD38+ HLA-DR+), regulatory (CD4+CD25+
CD127dim), HIV-specific, and CMV-specific T cells in a large
cohort of HIV controllers. We compared these data to those
observed in three well characterized control populations: HIV-
infected individuals with treatment-mediated viral suppression,
untreated HIV-infected ‘‘non-controllers’’ with high levels of
viremia, and HIV-uninfected individuals.
Characteristics of participants
A total of 52 HIV controllers with plasma HIV RNA levels ,75
copies/ml in the absence of antiretroviral therapy, 176 ART-
suppressed participants, 72 untreated HIV-infected non-controllers
with plasma HIV RNA levels .10,000 copies/ml, and 38 HIV-
uninfected participants contributed to these studies. Most were men
between 40 and 50 years of age, although compared to other HIV-
infected groups, HIV controllers were more likely to be women
(P=0.006, Table 1). The HIV controllers were also much more
likely to be hepatitis C virus (HCV) sero-positive than the other
HIV-infected groups (70% vs. 38%, P,0.001). While all HIV
controllers had plasma HIV RNA levels ,75 copies/ml, 19 (37%)
had an episode of a clinically measurable plasma HIV RNA level
.75 copies/ml in the previous year. While the HIV controllers had
significantly higher median CD4+ T cell counts than the ART-
suppressed (683 vs. 449 cells/mm3, P,0.001) and the non-
controllers (683 vs. 251 cells/mm3, P,0.001), 9 HIV controllers
theclinical definitionofAIDS (onewith Kaposi’ssarcomaand three
with CD4+ T cell counts persistently ,200 cells/mm3) despite
maintaining viral suppression in the absence of therapy.
HIV controllers have low Treg frequencies despite higher
T cell activation
We and others have previously reported that most HIV
controllers maintain strikingly high frequencies of CD4+ and
CD8+ T cells producing interferon (IFN)-c and interleukin (IL)-2 in
response to HIV Gag peptides [14,19,22], consistent with their
potential role in the control of viral replication. However, as our
group has recently reported in a smaller subset of participants
(n=30) , HIV controllers also had significantly higher
frequencies of activated (CD38+ HLA-DR+) CD8+ T cells
(Figure 1A) and CD4+ T cells (Figure 1B) than HIV-uninfected
participants (P,0.001 for both), even when restricting to HCV-
uninfected individuals (P,0.001). HIV controllers also had higher
frequencies of activated CD8+ T cells than ART-suppressed
participants (P=0.017), even after adjustment for HCV sero-status,
CD4+ T cell count, and gender (P=0.056). As we have previously
reported , higher frequencies of activated CD4+ and CD8+ T
cells were associated with greater CD4+ T cell depletion in HIV
controllers (P,0.001 for both, Figures S1A and S1B).
We hypothesized that a low Treg response to HIV infection
might explain why most HIV controllers maintain high HIV-
specific T cell responses but also high generalized T cell activation
levels. To assess this possibility, we sampled cryopreserved
peripheral blood mononuclear cells (PBMC) from 20 HIV
controllers, 20 ART-suppressed, and 20 untreated non-controllers,
and 34 healthy HIV–uninfected controls and compared the
frequencies of CD25+CD127dimCD4+ Tregs between groups.
Despite having higher frequencies of activated CD4+ and CD8+ T
cells than HIV-uninfected controls, the HIV controllers had a lower
median frequency of Tregs (3.9% vs. 4.9%, P=0.014, Figure 1C).
The HIV controllers also had a lower median frequency of Tregs
than the ART-suppressed (3.9% vs. 5.0%, P=0.008) and non-
controllers (3.9% vs. 6.8%, P,0.001). While there was no evidence
for a difference in Treg frequencies by gender within either group,
among both HIV-uninfected and HIV-infected individuals, lower
CD4+ T cell counts were associated with higher frequencies of
regulatory T cells (rho: -0.60, P,0.001). To account for differences
in absolute CD4+ T cell counts,we compared absolute regulatoryT
cell counts between groups. While absolute regulatory T cell counts
were similar between HIV-infected groups, the HIV controllers
continued to have a lower median CD25+CD127dimregulatory
CD4+ T cell count than HIV-uninfected participants (33 vs. 40
Table 1. Characteristics of Participants Contributing to T Cell Activation and HIV-specific T Cell Response Analyses.
VL, ,75 copies/ml
VL, ,75 copies/ml
Age, years43 (37 to 42)48 (45 to 52)46 (41 to 52)44 (40 to 49)
Female gender, no. (%)8 (22) 16 (31) 28 (16)12 (17)
CD4 count, cells/mm3
-683 (466 to 942) 449 (302 to 652) 251 (169 to 395)
Plasma HIV RNA level, log10copies/ml-
,1.94.5 (4.2 to 4.9)
Hepatitis C seropositive, no. (%)-24 (71)1
47 (27)23 (36)
Duration of HIV Diagnosis, years- 16 (10 to 19)13 (8 to 17)13 (9 to 16)
VL, Plasma HIV RNA Level.
1Hepatitis C virus serology was unavailable for 18 of 52 controllers.
Tregs and T Cell Activation in HIV Controllers
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It is surprising that HIV controllers have lower Treg frequencies
and counts than HIV-uninfected individuals since higher levels of
antigen stimulation and inflammation would be expected to cause
greater expansion of Tregs . Supporting this hypothesis,
higher plasma HIV RNA levels were strongly associated with
higher frequencies of Tregs among HIV-infected non-controllers
(rho: 0.72, P,0.001). Furthermore, among HIV controllers,
higher frequencies of regulatory T cells were associated with
higher frequencies of activated CD4+ T cells (rho: 0.49, P=0.03)
and activated CD8+ T cells (rho: 0.46, P=0.04, Figure 1D). Based
on these latter observations, we would have expected to observe
higher Treg frequencies in HIV controllers than in HIV-
uninfected individuals as a consequence of greater antigen
stimulation and T cell activation. The observation that HIV
controllers actually have lower Treg frequencies than HIV-
uninfected individuals thus suggests that HIV controllers have
an unusually weak Treg response to HIV infection, potentially
contributing to the high HIV-specific T cell responses and
generalized T cell activation observed.
Strong relationship between adaptive HIV-specific
immune response and generalized T cell activation in HIV
Since unusually low Treg responses in HIV controllers might
allow for both stronger adaptive HIV-specific immune responses
and generalized T cell activation, we hypothesized that there
would be a strong relationship between these two latter factors.
Among HIV controllers, higher frequencies of CD4+ T cells
producing both IFN-c and IL-2 in response to stimulation with
HIV Gag peptides were strongly associated with higher frequen-
cies of activated CD4+ T cells (rho: 0.36, P=0.012) and activated
CD8+ T cells (rho: 0.55, P,0.001, Figure 2A). Higher frequencies
of HIV Env-specific CD4+ T cell responses were also associated
with higher frequencies of activated CD8+ T cells (n=28,
P=0.46, P=0.014, Figure 2B). However, there was no evidence
for a relationship between Pol-specific or Nef-specific CD4+ T cell
responses and the frequency of activated CD4+ or CD8+ T cells.
HIV controllers with higher plasma HIV-specific antibody levels
(as assessed by de-tuned ELISA) also had higher frequencies of
Figure 1. HIV Controllers Have Abnormally Low Treg Frequencies Despite Abnormally High T Cell Activation. The frequency of
activated (CD38+ HLA-DR+) CD8+ T cells (A) and CD4+ T cells (B) in fresh whole blood was compared between 52 HIV-infected untreated HIV
controllers, 37 HIV-uninfected participants, 176 HIV-infected participants with plasma HIV RNA levels ,75 copies/ml on antiretroviral therapy, and 64
untreated HIV-infected participants with plasma HIV RNA levels .10,000 copies/ml. Cryopreserved PBMC from 34 healthy HIV-uninfected participants
in ACTG 5015 (HIV-), 20 HIV controllers, 20 antiretroviral therapy (ART)-treated participants with plasma HIV RNA levels ,75 copies/ml and 20
untreated HIV-infected participants with plasma HIV RNA levels .10,000 copies/ml were also evaluated for the frequency of CD4+ Tregs
(CD25+CD127dim). PBMC preparations were first gated on lymphocytes based on their forward and side scatter properties, then gated for CD4+
lymphocytes, then CD4+ lymphocytes positive for CD25 and only dimly expressing CD127, results expressed as a percentage of the parent CD4+
population (C). HIV controllers had lower frequencies of Tregs than HIV-uninfected controls and both other HIV-infected groups (D). Among HIV
controllers, higher frequencies of activated CD8+ T cells were associated with higher frequencies of Tregs (E). The curve represents the best-fit linear
Tregs and T Cell Activation in HIV Controllers
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activated CD4+ T cells (rho: 0.46, P=0.025) and CD8+ T cells
(rho: 0.60, P=0.002, Figure 2D).
The frequency of HIV-specific CD8+ T cells were less consistently
associated with the frequency of activated T cells. In general, there
was little evidence for an association between the frequency of HIV-
specific CD8+ T cells producing both IFN-c and IL-2 and the
frequency of activated CD4+ or CD8+ T cells. However, higher
frequencies of activated CD8+ T cells tended to be associated with
higher frequencies of CD8+ T cells producing IFN-c but not IL-2 in
response to HIV Nef (rho: 0.42, P=0.025, Figure 2C), Pol (rho: 0.39,
P=0.045), and Gag peptides (rho:0.22, P=0.14).
HIV controllers also have high CMV-specific CD4+ T cell
We next hypothesized that an unusually low Treg response in
HIV controllers might also contribute to higher adaptive immune
responses directed at other chronic viral infections. We chose to
focus on cytomegalovirus (CMV) since CMV is nearly ubiquitous
in HIV infected individuals, is typically controlled to nearly
undetectable levels in individuals with intact immune systems, yet
elicits high frequencies of CMV-specific T cells even in HIV-
uninfected individuals [37,38]. To address this, we compared
CMV-specific T cell responses between HIV-uninfected but
CMV-sero-positive controls, HIV controllers, and untreated
HIV-infected participants with varying plasma HIV RNA levels
(75–2000, 2001–10,000, and .10,000 copies/ml). The HIV
controllers had higher CMV pp65-specific IFN-bright CD4+ T
cell responses than HIV-uninfected controls (P,0.001, Figure 3A).
While HIV controllers had similar frequencies of pp65-specific
IFN-bright CD4+ T cells as untreated HIV-infected participants
maintaining low but detectable plasma HIV RNA levels between
75 and 2,000 copies/ml, they had significantly higher frequencies
Figure 2. Relationship between Adaptive HIV-specific Immune Responses and CD8+ + T Cell Activation in HIV Controllers. The
association between the frequency of activated (CD38+ HLA-DR+) CD8+ T cells and the frequency of CD4+ T cells producing both IFN-c and IL-2 after
stimulation with overlapping HIV Gag (A) or HIV Env peptides (B), CD8+ T cells producing only IFN-c after stimulation with overlapping Nef peptides
(C), and plasma HIV-specific antibody levels (as assessed by de-tuned ELISA, D) were assessed among HIV Controllers. The curves in each plot
represent best-fit linear or quadratic regression models using untransformed data.
Tregs and T Cell Activation in HIV Controllers
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of pp65-specific IFN-bright CD4+ T cells than HIV-infected
participants with plasma HIV RNA levels .10,000 copies/ml
(P=0.003). Across all 4 groups of untreated HIV-infected
participants, lower plasma HIV RNA levels were associated with
higher pp65-specific CD4+ T cell frequencies (P=0.001). Even
after adjustment for age, HIV controllers continued to have higher
pp65-specific CD4+ T cell responses than HIV-uninfected
participants (P=0.003) and untreated HIV-infected participants
with plasma HIV RNA levels .10,000 copies/ml (P=0.016).
Notably, HIV controllers with the highest frequencies of pp65-
specific CD4+ T cells also had the highest frequencies of Gag-
specific CD4+ T cells (rho: 0.32, P=0.024, Figure 3B). Similar
trends were observed when comparing the frequency of CMV-
specific IFN-c+ IL-2+ CD4+ T cells across groups in a smaller
subset of individuals (data not shown). There was no evidence for a
consistent relationship between pp65-specific CD8+ T cell
responses and plasma HIV RNA levels among untreated HIV-
A wealth of data now suggest that most HIV controllers
maintain control of viral replication at least in part through potent
HIV-specific T cell responses [6,7,8,9,10,11,12,13,14,20,22],
observations that have spurred the development of vaccines that
elicit T cell responses against HIV. However, the mechanisms
responsible for a strong HIV-specific T cell response in HIV
controllers may not be without important consequences for the
immune system. As our group recently reported, most HIV
controllers have abnormally high levels of immune activation,
which is associated with significant CD4+ T cell depletion and
even AIDS despite continued control of virus replication . In
the current study, we have expanded upon this prior work and
assessed potential mechanisms to explain this paradox. First,
despite abnormally high T cell activation levels, HIV controllers
have significantly lower Treg frequencies than HIV-uninfected
individuals. Second, we observed a strikingly strong relationship
between adaptive HIV-specific CD4+ T cell and antibody
responses and generalized T cell activation in HIV controllers.
Third, we observed unusually high CMV-specific CD4+ T cell
responses in HIV controllers, suggesting that their ability to mount
strong T cell responses to chronic viral infections may not be
specific for HIV. Collectively, these observations suggest that a low
Treg response may allow some HIV controllers to maintain viral
control with a strong cytotoxic HIV-specific T cell response, but
might also contribute to the negative inflammatory consequences
of generalized T cell activation in this setting (Figure 4).
Multiple mechanisms have been proposed to explain why HIV
controllers maintain low to undetectable levels of viral replication in
the absence of therapy. While it is possible that some HIV
controllers may simply be infected with defective viruses , most
harbor replication competent viruses that lack gross deletions or
lethal mutations [40,41]. Several lines of evidence suggest an
important role of HIV-specific T cells in the control of viral
replication. For example, most HIV controllers maintain unusually
high frequencies of HIV-specific CD4+ and CD8+ T cells
[6,7,8,9,10,11,12,13,14,19], as well as HIV-specific CD8+ T cells
with greater proliferative and cytotoxic potential [8,12,42]. While
strong HIV-specific T cell responses could conceivably be a
consequence of poor viral fitness [43,44], HIV controllers are
highly enriched for protective class I HLA alleles (i.e., B5701) and
polymorphisms associated with HLA C expression [15,16,17,18],
suggesting that CD8+ T cell responses may play an important role
in the control of HIV replication. Some HIV controllers also have
high frequencies of CD4+ T cells with cytotoxic activity [45,46].
However, many HIV controllers lack a protective HLA type, have
very low frequencies of HIV-specific T cells, or maintain control of
viral replicationeven after documented escape from HLA-restricted
Figure 3. HIV Controllers Have High Frequencies of CMV-specific T Cells. (A) The frequency of CD4+ T cells producing IFN-c after incubation
with CMV pp65 peptides in vitro (representative flow plot depicted in Figure 1 from reference ) was assessed in HIV-uninfected but CMV-
seropositive controls, HIV controllers, and untreated HIV-infected participants with varying degrees of detectable viremia (75–2000, 2001–10,000, and
.10,000 copies/ml). The HIV controllers had higher CMV pp65-specific IFN-bright CD4+ T cell responses than HIV-uninfected controls and HIV-
infected participants with high levels of viremia. HIV controllers with higher CMV pp65-specific CD4+ T cell responses also had higher HIV Gag-specific
CD4+ T cell responses (curve represents linear regression model on untransformed data, B).
Tregs and T Cell Activation in HIV Controllers
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epitopes [14,20,21,22]. In these individuals, non-T cell-mediated
mechanisms of control are likely. For example, HIV controllers are
highly enriched for HLA and KIR allotypes associated with
enhanced natural killer cell responses [47,48]. Other immunologic
mechanisms and host restriction factors that are yet to be fully
characterized are also likely to play a role [16,49].
It is important to acknowledge this heterogeneity in the
mechanisms of viral control in HIV controllers as some
mechanisms are likely to be associated with more negative
inflammatory consequences than others. While other cohorts have
not observed increased T cell activation levels in HIV controllers
[50,51], these studies either included individuals with nef-deleted
viruses or only included HIV controllers maintaining normal
CD4+ T cell counts. When selecting HIV controllers solely on the
basis of their ability to control viral replication, it is clear that some
controllers eventually progress to significant levels of CD4+ T cell
depletion [5,52,53], and these individuals have the highest T cell
activation levels . In the current study, we also observed that
HIV controllers with the highest HIV-specific CD4+ T cell
frequencies and antibody levels had the highest levels of
generalized T cell activation and the greatest degree of CD4+ T
cell depletion. Thus, the HIV-specific immune response and
generalized T cell activation are tightly linked in HIV controllers
and these relationships appear to be stronger than those observed
in untreated HIV-infected individuals with high levels of viral
replication [54,55]. While we cannot exclude the possibility that
higher adaptive immune responses are simply a consequence of
greater degrees of low-level viral replication - particularly in
lymphoid tissues, differences between HIV controllers in the
degree of adaptive immune responses and T cell activation may
well reflect host differences in the immune response elicited by any
given level of virus replication. The extent of microbial
translocation may be one factor modulating the response to low-
level HIV replication. As we reported previously, most HIV
controllers have abnormally high plasma lipopolysaccharide levels
, which might drive generalized immune activation, but also
serve as an adjuvant for HIV-specific T cell responses, particularly
in gut-associated lymphoid tissue where the majority of HIV
replication is thought to occur.
Alternatively, HIV controllers may be enriched for host genetic
factors associated with strong innate and/or weak Treg responses
to viral infection. Indeed, we found that HIV controllers had
significantly lower frequencies of CD25+CD127dimCD4+ Tregs
in peripheral blood than HIV-uninfected individuals despite much
higher levels of T cell activation. While we cannot exclude the
possibility that HIV controllers preferentially retain Tregs in
lymphoid tissues, a recent study also found low frequencies of
Tregs in tissues of HIV controllers . While the specific
mechanisms mediating the unusually low Treg frequencies in HIV
controllers remain unclear, a low Treg response is likely to have
competing effects in this setting. For example, several studies have
argued that that these cells are detrimental in HIV infection by
inhibiting HIV-specific T cell responses [56,57,58,59,60,61], while
others have argued that these cells are beneficial by reducing
generalized T cell activation [62,63,64,65]. Inferring causal
relationships is particularly challenging in cross-sectional studies
of in vivo Treg frequency in HIV-infected individuals since Tregs
may be induced and expanded by viral replication and resultant
inflammation , but once induced, act to decrease inflamma-
tion. Accordingly, we observed that HIV controllers with higher
levels of immune activation had higher frequencies of Tregs,
suggesting that inflammation was driving the induction of Tregs.
However, HIV controllers had lower Treg frequencies than HIV-
uninfected individuals despite having much higher T cell
activation, suggesting a strikingly low Treg response for the
degree of immune activation observed. This unusually low Treg
response in HIV controllers is therefore likely to be a significant
contributor to the high generalized T cell activation and HIV-
specific T cell responses observed. These results are consistent with
a recent report of decreased inhibitory immunoregulatory receptor
CTLA-4 expression on CD4+ T cells in HIV controllers .
Our results differ from another recent report describing
preserved Treg frequencies (as defined by FoxP3 expression) in
the peripheral blood of a much smaller cohort of 12 HIV
controllers . However, FoxP3 can be expressed early in the
activation of effector CD4+ T cells without any regulatory
function [67,68,69,70,71,72], so the preserved FoxP3 expression
described in that study may simply reflect the presence of recently
Figure 4. Theoretical Model to Describe Positive and Negative Consequences of Low Treg Frequencies in HIV Controllers. A
theoretical model to describe the potential positive and negative consequences of low Treg frequencies in HIV controllers is presented. While a low
Treg response might increase HIV-specific T cell responses, contributing to the clearance of HIV-infected cells and the maintenance of extremely low
levels of viral replication, a low Treg response might also increase generalized T cell activation, contributing to CD4+ T cell decline and other
inflammation-associated comorbidities even in the presence of very low levels of viral replication.
Tregs and T Cell Activation in HIV Controllers
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activated effector CD4+ T cells, particularly since the co-
expression of CD25 and FoxP3 in CD4+ T cells was not
presented. Low expression of CD127, as measured in our study,
may help distinguish Tregs from activated T cells and is now
routinely used with CD25 to quantify the frequency of Tregs with
suppressor function [73,74,75]. It should be noted that among
HIV-infected individuals with high levels of viral replication,
gating on CD4+/CD25+/CD127dimmay include some cells that
do not express FoxP3 and thereby lack regulatory function .
However, Treg frequencies defined by CD4+/CD25+/CD127dim
and CD4+/CD25hi/FoxP3+ are highly correlated in HIV-infected
individuals with undetectable plasma HIV RNA levels (r=0.91,
P,0.001) . Thus, the low frequency of CD4+/CD25+/
CD127dimcells we observed in HIV controllers relative to HIV-
uninfected controls and ART-suppressed individuals (all groups
with undetectable viremia) almost certainly reflects a low
frequency of Tregs in HIV controllers. Lastly, even if HIV
controllers had similar levels of Tregs to HIV-uninfected
individuals as has been suggested in another recent report using
HIV controller samples from the same cohort , they would still
have unusually low Treg frequencies relative to the expansion of
activated T cells observed.
Consistent with the hypothesis that HIV controllers are
predisposed to a weak Treg response to chronic viral infections,
we observed significantly higher CMV-specific CD4+ T cell
responses in HIV controllers than non-controllers and HIV-
uninfected individuals. While we cannot exclude the possibility
that greater CMV shedding explains the higher CMV-specific
CD4+ T cell responses in HIV controllers, CMV shedding tends
to be lower in individuals with higher CD4+ T cell counts and
lower plasma HIV RNA levels . Thus, the expansion of CMV-
specific CD4+ T cells in HIV controllers is unlikely to be driven by
higher levels of antigen and is more likely to reflect a more robust
proliferation of CD4+ T cells in response to CMV infection. HIV
controllers co-infected with hepatitis C virus (HCV) might also
exhibit stronger HCV-specific responses than individuals with
higher levels of HIV replication . While lower levels of HIV
replication may allow for preservation of antigen-specific immune
responses, the high CMV-specific CD4+ T cell frequency in HIV
controllers relative to HIV-uninfected CMV-seropositive individ-
uals cannot be explained by this mechanism alone. While another
recent report suggested that HLA B5701+ elite controllers
maintain similar CMV- and HCV-specific CD8+ T cell responses
as non-controllers, CD4+ T cell responses were not assessed in that
study , and epidemiologic data suggest that HIV controllers
are much more likely to spontaneously clear HCV than viremic
HIV-infected individuals and HIV-uninfected individuals infected
with HCV .
In summary, we have observed that while most elite
controllers maintain high HIV-specific T cell responses, most
also have abnormally high generalized T cell activation levels,
which may occasionally contribute to significant CD4 depletion
even in the absence of clinically detectable viremia. Further-
more, those with the highest HIV-specific T cell responses have
the highest levels of generalized immune activation, suggesting
possible inflammatory consequences of T cell-mediated control
of HIV replication. An unusually low regulatory T cell response
to HIV infection may well explain this phenomenon. Perhaps the
best immune response to HIV infection is one that maintains
control of viral replication while minimizing negative inflamma-
tory consequences. Some elite controllers are able to maintain
this balance and understanding the mechanisms of control in
these individuals is likely to have important implications for HIV
Materials and Methods
For comparison of HIV-specific immune responses and T
cell activation levels.
HIV-infected adults were sampled from
the Study of the Consequences of the Protease Inhibitor Era
(SCOPE), a clinic-based cohort of over 1000 chronically HIV-
infected individuals at the University of California San Francisco.
From this cohort, we evaluated three distinct groups of HIV-
infected individuals: (1) HIV controllers, defined as HIV-
seropositive individuals maintaining plasma HIV RNA levels
,75 copies/ml in the absence of therapy (episodes of clinically
detectable viremia in the previous year were allowed if they were
individuals maintaining plasma HIV RNA levels ,75 copies/ml
on antiretroviral therapy; and (3) untreated HIV ‘‘non-controllers’’
with plasma HIV RNA levels above 10,000 copies/mL. T cell
activation data have been previously reported on 30 of the 52 HIV
controllers and all of the ART-suppressed and untreated patients
in the current report , HIV-specific T cell response data have
also been reported on these individuals recently . HIV-
uninfected individuals were also sampled from a study of the
immunologic determinants of atherosclerosis and have been
reported on previously [14,82].
For comparisons of CMV-specific T cell responses
In addition to the above participants,
untreated HIV-infected participants with plasma HIV RNA
levels between 75 and 10,000 copies/ml were sampled from the
SCOPE cohort. HIV-negative individuals were also sampled from
a trial of post-exposure prophylaxis following a non-occupational
exposure to HIV . Only CMV-seropositive HIV-negative
participants were included in the analyses of CMV-specific T cell
For comparison of Tregs between groups.
PBMC availability, cryopreserved PBMC from different SCOPE
participant-timepoints were sampled for the measurement of both
Treg frequency and T cell activation levels in 20 HIV controllers,
20 HAART-suppressed participants, and 20 non-controllers. Only
specimens on participants with CD4+ T cell counts .350 cells/
mm3were selected for these analyses to ensure adequate overlap
between groups. For the Treg analyses, cryopreserved PBMC
were also sampled from 34 healthy HIV-uninfected controls from
the AIDS Clinical Trials Group 5015 study .
All participants provided written informed consent and this
research was approved by the institutional review board of the
University of California, San Francisco.
T cell activation.
whole blood was analyzed by four-color flow cytometry on a
Beckman Coulter Epics XL flow cytometer. Blood was stained on
a Beckman Coulter Prep Plus and lysed on a Beckman Coulter
TQ Prep. Activated (CD38+/HLA-DR+) T cells were identified
with FITC-conjugated anti-HLA-DR, PE-conjugated anti-CD38
(both from BD Bioscience), PC5-conjugated anti-CD3 and PE-
texas red conjugated anti-CD4 or CD8 (Beckman Coulter). The
activation markers CD38 and HLA-DR were gated from the
CD3+CD4+ or CD3+CD8+ cells on a 2-dimensional dot plot
where quadrant gates, set on an isotype control, were used to
define positive and negative populations. T cell activation levels
were reported as the percentage of CD4+ and CD8+ T cells
expressing both HLA-DR and CD38.
Freshly collected, EDTA-anticoagulated
Tregs and T Cell Activation in HIV Controllers
PLoS ONE | www.plosone.org7 January 2011 | Volume 6 | Issue 1 | e15924
Cytokine flow cytometry.
overlapping by 11 amino acids) of the HIV-1 p55 Gag, Pol, Nef,
Env, or CMV pp65 protein (BD Biosciences, San Jose, CA) for 6 h
in the presence of brefeldin A, as reported recently .
Unstimulated cells and superantigen staphylococcal enterotoxin
B (Sigma Aldrich)-stimulated cells were used as negative and
positive controls, respectively. Cells were fixed, permeabilized, and
stained with FITC- conjugated anti-interferon (IFN)-c, PE-
Bioscience) and PC5-conjugated anti-CD4 (Beckman Coulter)
and data was collected on a Becton Dickinson FACSCalibur. The
fractions of CD4+ and CD8+ T cells secreting IFN-c and/or IL-2
were determined using FlowJo software (TreeStar). In our primary
analysis of CMV-specific T cell responses, we focused on cells that
stained brightly for IFN-c. The ‘‘IFN-c bright’’ gate was set 3
decades above the IFN-c-negative population in non-stimulated
control, as previously described (representative flow plot depicted
in Figure 1 from reference ). Cells were initially defined as
lymphocytes based on forward- and side-scatter profiles. CD4+
and CD8+ anchor gates were drawn on the CD3+CD4+ and
CD3+CD4- populations, respectively. At least 10,000 CD3+CD4+
and CD3+CD4- events were collected for the majority of subjects;
data were excluded if ,4,000 events were collected. Cytokine
secretion levels in the negative control were subtracted to correct
for nonspecific cytokine secretion.
Cryropreserved PBMC were evaluated
using 4-color flow cytometry. Mouse anti-human monoclonal
antibodies (CD4, CD8, CD25, CD45RO, and CD127) conjugated
to fluorescein isothiocyanate (FITC), phycoerythrin (PE), PerCP,
and allophycocyanin (APC) from BD Biosciences (San Jose, CA) or
Coulter Immunology (Miami, FL) were used to stain the PBMC
preparations. Non-specific antibody binding to Fc receptors was
blocked by pre-incubation of the cells with Fcc-receptor block
(Miltenyi Biotec, Auburn, CA). All samples were evaluated within
24-hours of staining using a FACSCaliburTMflow cytometer.
Logical gating was used to identify the frequency of T regulatory
(CD4+/CD25+/CD127dim) T lymphocyte populations (Figure 1B)
[73,74,75]. Results are expressed as the percentage of the parent
CD4+ T cell population.
HIV Antibody Levels.
A ‘‘de-tuned’’ enzyme immunoassay
(Organon Tecnika Vironostika [OTV], BioMerieux) was used to
measure semiquantitative HIV antibody levels on a subset of HIV
Fresh whole blood was stimulated
pools (15-amino-acidpeptide peptides
controllers . The OTV is a second-generation ELISA that
detects both IgG and IgM antibodies to HIV-1 and is FDA-
approved for diagnostic testing. The less sensitive modification
involves testing 1:20,000 dilutions of plasma under abbreviated
incubation conditions and calculating a standardized optical
density (SOD) for each sample .
Continuous variables were compared
between groups with Kruskal Wallis tests followed by Wilcoxon
ranksum tests for pairwise comparisons. Dichotomous variables
were compared between groups with chi square and Fisher’s exact
tests. Relationships between continuous variables were assessed
with Spearman’s rank order correlation coefficients. Adjusted
differences between groups were assessed with linear regression,
calculating standard errors with heteroskedasticity-consistent
covariance matrix estimators and log-transforming outcomes
when necessary to satisfy model assumptions .
The authors would like to thank Dr. Dennis Hartigan-O’Connor for his
thoughtful discussion of this work.
Formulated the hypotheses, contributed to the design of the research,
analyzed the data, interpreted the results, and wrote the manuscript: PWH.
Contributed to framing the hypotheses, the design of the research,
supervised the Treg measurements, and provided access to ACTG control
samples: ALL. Performed the T cell activation and cytokine flow cytometry
measurements: ES. Performed the Treg measurements: JAM CB. Provided
access to the low-level viremia and HIV-specific antibody measurements,
analyzed these data, and contributed to the interpretation of these
measures: HH. Provided access to the cytokine flow cytometry data and
contributed to the interpretation of these measures: BE. Supervised the
HIV-specific antibody measurements and contributed to the interpretation
of these results: PJN MPB. Provided access to patient samples and
contributed to the design and analysis of the data: JNM. Contributed to
framing the hypotheses and interpreting the results: JMM. Provided patient
samples and contributed to framing the hypotheses, designing the research,
and interpreting the results: SGD.
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