Suppression of HIV-specific T cell activity
by lymph node CD25?regulatory T cells
from HIV-infected individuals
Audrey Kinter*, Jonathan McNally, Lindsey Riggin, Robert Jackson, Gregg Roby, and Anthony S. Fauci*
Laboratory of Immunoregulation, National Institute of Allergy and Infectious Diseases, National Institutes of Health, 9000 Rockville Pike,
Bethesda, MD 20892
Contributed by Anthony S. Fauci, December 29, 2006 (sent for review November 29, 2006)
CD25?CD4?FoxP3?regulatory T (Treg) cells isolated from the
peripheral blood of asymptomatic HIV-infected individuals have
been demonstrated to significantly suppress HIV-specific immune
responses in vitro. CD25?Treg cell suppressor activity in the
peripheral blood seems to diminish with progression of HIV dis-
ease, and it has been suggested that loss of Treg cells contributes
to aberrant immune activation and disease progression. However,
phenotypic studies suggest that Treg cells may migrate to, and be
maintained or even expanded in, tissue sites of HIV replication.
Currently, it is not known whether tissue-associated Treg cells
maintain suppressive activity in the context of HIV infection,
particularly in individuals with advanced disease. The present
study demonstrates that CD25?Treg cells isolated from lymph
nodes and peripheral blood of HIV?subjects, even those with high
viral loads and/or low CD4?T cell counts, maintain potent sup-
pressive activity against HIV-specific cytolytic T cell function. This
activity was better in lymph node as compared with peripheral
blood, particularly in patients with high levels of plasma viremia.
In addition, the expression of certain CD25?Treg-associated mark-
from those on CD4?T cell subsets isolated from the peripheral
blood. These data suggest that CD25?Treg cell-mediated suppres-
sion of HIV-specific responses continues throughout the course of
HIV-specific CTL activity in lymphoid tissue, may considerably
impact the ability to control HIV replication in vivo.
cytolytic T lymphocyte ? cytokine ? proliferation
tion to limit potential immune-mediated damage to the host.
Among the best characterized of these mechanisms are
CD25?FoxP3?regulatory T (Treg) cells, a suppressor CD4?T
cell subset initially described as playing a critical role in con-
taining certain types of autoimmunity in animal models (1). A
potentially deleterious consequence of CD25?Treg cells is the
suppression of appropriate antigen-specific immune responses.
In this regard, CD25?Treg cells have been implicated in playing
a role in disease/pathogen persistence, such as certain solid
tumors and parasitic and bacterial infections (2–8). Further-
more, CD25?Treg cells accumulate and expand at sites of
antigen expression/ inflammation as well as in draining lymph
nodes where they seem to exert particularly strong site-localized
immunosuppression (2, 4, 9).
Chronic HIV infection is characterized by loss of CD4?T
cells, a broad array of immune dysfunctions, and persistent
immune activation (10–12). Whereas the direct and indirect
roles of HIV and its gene products in immune dysfunction are
well documented (13, 14), recent evidence suggests that normal
host-mediated negative immunoregulatory mechanisms, trig-
gered by persistent antigenimia and immune activation, may also
impact the immune competence of infected individuals (15). In
this regard, numerous in vitro studies have demonstrated that
nder conditions of persistent antigen exposure and immune
activation numerous immunosuppressive mechanisms func-
CD25?Treg cells isolated from the peripheral blood of asymp-
tomatic HIV-infected individuals significantly suppress HIV-
specific CD4?and CD8?T cell immune responses (16–18).
However, progressive HIV disease has been associated with
reduced frequencies or suppressor activity of CD25?Treg cells
in the peripheral blood (PB) (17, 19–21). These observations
have led to the hypothesis that HIV infection is associated with
a selective loss of CD25?CD4?Treg cells leading to a reduced
ability to control HIV-associated aberrant immune activation
and associated immune dysfunctions, ultimately resulting in
more rapid disease progression. An alternative hypothesis is that
CD25?Treg cells are redistributed to tissue sites of HIV
expression (19, 22–24). Currently, the data supporting this later.
hypothesis is largely phenotypic and it is not known whether
tissue-associated Treg cells, particularly in individuals with ad-
vanced disease, maintain their ability to suppress HIV-specific
immune responses. Localization to and perhaps activation of
CD25?Treg cells at lymphoid tissue sites might have a partic-
ularly negative impact on the ability of HIV-specific immune
responses to control viral replication and spread. The present
study investigates the suppressive activity of CD25?Treg cells
isolated from lymph nodes (LN) and PB of chronically HIV-
infected individuals at various stages of disease progression,
including those with high viremia and/or CD4?T cells ?150/?l.
Phenotypic Analyses of CD25?Treg Cells Isolated from Lymphoid
Tissue and Peripheral Blood.TotalPBmononuclearcells(MC)and
LNMC were stained for CD4, CD25, intracellular FoxP3 and
additional putative CD25?Treg markers (CD127, SCTLA-4, or
GITR). Previous studies with human PBMC have clearly cor-
related suppressor activity with a CD25hiphenotype (25) and
therefore, in the present study, CD25?Treg cells were identified
by CD25hi[mean fluorescence intensity (MFI) ? 70] and FoxP3
(26, 27) coexpression (Fig. 1A). The purity of the CD4?T cell
subsets that were isolated on the basis of ?CD25hi expression
and subsequently added back to various cultures in functional
assays (see below) is illustrated in Fig. 1B. Comparing matched
LNMC and PBMC (both obtained within 24 h) isolated from 11
HIV-infected subjects (Table 1), several significant differences
Author contributions: A.K. and A.S.F. designed research; J.M., L.R., and R.J. performed
research; G.R. contributed new reagents/analytic tools; G.R. coordinated patient recruit-
ment and procedures; and A.K. and A.S.F. wrote the paper.
The authors declare no conflict of interest.
Abbreviations: Treg, regulatory T; LN, lymph node; PB, peripheral blood; MFI, mean
fluorescence intensity; MC, mononuclear cell; VL, viral load.
*To whom correspondence should be addressed at: Laboratory of Immunoregulation,
National Institute of Allergy and Infectious Diseases, National Institutes of Health, Build-
ing 10, Room 6A33, 10 Center Drive, Bethesda, MD 20892. E-mail: email@example.com
This article contains supporting information online at www.pnas.org/cgi/content/full/
February 27, 2007 ?
vol. 104 ?
the frequency of CD25hiFoxP3?Treg cells was higher in LNMC
than in PBMC (Fig. 1A and Fig. 2 Top plot). An increased
frequency of CD25hiFoxP3?CD4?Treg cells in LNMC versus
PBMC was observed in all subjects regardless of the level of
plasma viremia (data not shown). Second, the intensity of FoxP3
expression (MFI) was significantly higher in CD25?FoxP3?
Tregs cells from LN compared with PB (Figs. 1C and 2 Bottom
plot). There was no significant correlation between plasma
viremia or CD4?T cell count and CD25hiFoxP3?Treg fre-
quency or FoxP3 MFI in either the PB or LN (data not shown).
Finally, as reported (19), the frequency of CD25?CD4?T cells
expressing FoxP3 was significantly greater in LNMC than in
PBMC (Fig. 2 Middle plot).
Several surface(s) markers have been reported to be differen-
tially expressed on CD25?Treg cells compared with normal CD4?
T cells in the peripheral blood. Human peripheral blood CD25hi
FoxP3?Treg cells are reported to be exclusively CD127lo/negand
enriched for sCTLA-4?and GITR?whereas normal resting
(CD25-FoxP3-) CD4?T cells are CD127hiand negative for
to adequately discriminate between CD25hiFoxP3?Treg cells and
normal resting (CD25-FoxP3-) CD4?T cells (identifying ?50% of
CD25hiFoxP3?subset) (Fig. 3 Middle and Bottom panels). There
was a significantly higher frequency of CD25hiFoxP3?CD4?T
cells expressing GITR in the LN compared with the PB (Fig. 3
Bottom panel). CD127 was the most clearly differentially expressed
marker with only 5–7% of the CD25hiFoxP3?CD4?T cell subset
in the peripheral blood expressing a CD127hi(MFI ? 40) pheno-
as clear-cut in LNMC where up to 30% of CD25hiFoxP3?CD4?
T cells expressed CD127hi(Fig. 3 Top panel). The expression of
sCTLA-4, GITR and CD127 on CD25?FoxP3?CD4?T cells is
shown in supporting information (SI) Fig. 7.
Functional Studies. In the present study, freshly isolated, matched
PBMC and LNMC of chronically HIV-infected subjects 1–5 (see
Table 1) were assessed for CD25?cell-mediated suppression of
HIV-specific T cell function. Two of the study subjects had
advanced disease (patients 1 and 5) with CD4?T cell counts/?l
of ?150; patient 5 had a viral load (VL) of 58,791 copies/ml
whereas patient 1 was on long-term antiretroviral therapy
(ART), had a VL ?50 copies/ml, and had Kaposi sarcoma. The
remaining patients had CD4?T cell counts of ?350 and variable
VL (?50–203,495 copies per ml). CD25?Treg activity was
assessed by comparing HIV-specific T cell function in unfrac-
tionated versus CD25?cell-depleted MC, unless otherwise
Lymphoproliferation Assay. The most common method to assess
CD25?Treg suppressor activity has been to demonstrate inhi-
bition of normal CD25?CD4?T cell proliferation. Using such
functional assays, we previously reported on the difficulty in
detecting suppressive activity of PB-derived CD25?Treg cells
isolated from chronically HIV-infected individuals with ad-
vanced disease compared with HIV?subjects with a favorable
clinical status (17). In the present study, total and CD25?hi
cell-depleted CD4?T cells (see Fig. 1B) isolated from PBMC
and LNMC of patients 1–5 (Table 1) were compared for
proliferation in response to HIV p24. As expected, CD4?T cells
from the two individuals with advanced disease (subjects 1 and
5) failed either to proliferate to HIV p24 or failed to exert
CD25? cell-mediated suppression (? 40% inhibition) (Fig. 4).
Although subject 1 had been on ART and had an undetectable
CD25hi CD4+ Tc
CD25– CD4+ Tc
Total CD4+ Tc
CD4?T cells of PBMC compared with LNMC. (A) Freshly isolated PBMC and
LNMC were stained for CD3, CD4, CD25 and intracellular FoxP3. CD25 and
FoxP3 expression was analyzed in the CD3?CD4?population. (A) CD25?Treg
cells in PBMC and LNMC were quantified as FoxP3?and CD25hi(MFI ?70) as
indicated in the box in the upper right quadrant. (B) Representative example
(C) Representative example of FoxP3 expression intensity (MFI) on CD25?
CD4?T cells in LN and PB.
Representative phenotypic analysis of CD25 and FoxP3 expression in
Percent of CD4+ T Cells
Percent of CD4+ T Cells
p = 0.0005
p = 0.003
p = 0.0009
within LNMC is significantly greater than within PBMC. CD25hi(Top) and
CD25?(Middle) FoxP3?CD4?T cell frequencies and FoxP3 MFI in CD25?CD4?
T cells (Bottom) in parallel LNMC and PBMC isolated from 11 chronically
The frequency and MFI of FoxP3 expression in CD4?T cell subsets
Kinter et al. PNAS ?
February 27, 2007 ?
vol. 104 ?
no. 9 ?
viral load for some time, both these individuals had ?150 CD4?
T cells per ?l, suggesting that CD4?T cell count was most
relevant to Treg activity in this assay. As reported (17), in vitro
CD25?Treg activity was most potent within both PBMC and
LNMC of the individual (subject 2) with the best clinical status
(CD4?T cells ?500/?l and VL ?50) (Fig. 4). The effect of
CD25?hicell depletion on allogeneic antigen-induced prolifer-
ation in LNMC and PBMC is shown in SI Fig. 8.
Assays of Cytolytic Activity. As HIV-specific proliferation assays
proved to be problematic for the assessment of CD25? Treg
activity, we sought to measure CD25? Treg suppressor capacity
using another relevant functional assay. Virus-specific CD8?T
cell-mediated cytolytic (CTL) activity is thought to be critical in
(30–34). As the lymphoid tissue is the major site of active HIV
replication (35–37), HIV-specific CTL activity in the lymph
nodes likely represents an important immunologic response with
regard to control of HIV replication in vivo. In the present study
we compared unfractionated and CD25?hicell-depleted PBMC
and LNMC from patients 1–5 for HIV-specific cytolytic activity
(Granzyme B substrate cleavage) in HIV Gag peptide restimu-
lation assays. In addition, with cells from patients 3–5, we were
able to assess relative Treg suppressive activity in CD25?MC
plus CD25?hiversus CD25?CD4?T cell add-back experiments.
As seen in a representative experiment (Fig. 5), HIV Gag-
specific target cell ‘‘killing’’ was enhanced in CD25?cell-
depleted (Middle plots) compared with unfractionated (Top
plots) LNMC and was dramatically suppressed by the readdition
of 30% CD25hi?CD8?(?85% CD4?T cells; data not shown)
Table 1. Clinical profiles of study subjects
PatientVL (HIV RNA copies per ml)
Tc/?l CD4:CD8 Tc ratioART
ART, antiretroviral therapy; Tc, T cells .
*VL measured using assays with a limit of detection of 10,000 HIV RNA copies per ml.
Percent of CD4+ T Cell Subset
CD25?FoxP3?and CD25hiFoxP3?CD4?LNMC and PBMC subsets were ana-
PBMC subsets. Data are of mean percent ? SD of data obtained from LNMC
and PBMC isolated from 11 chronically HIV-infected individuals.
Comparison of PB and LN for the expression of surface markers used
Percent Suppression of
HIV p24 Proliferation
and PBMC of subjects 1–5 were stimulated with HIV p24 protein for 6 days,
then pulsed with3H thymidine 16 h. Percent suppression of proliferation by
CD25?cells was determined by comparing net cpm obtained in total versus
CD25?cell-depleted CD4?T cell cultures (?40% was arbitrarily considered to
be a significant level of suppression). N.R., no response.
CD25? Treg suppressive capacity assessed in HIV p24-specific prolif-
www.pnas.org?cgi?doi?10.1073?pnas.0611423104Kinter et al.
LNMC to CD25?cell-depleted LNMC (Bottom Right plot).
HIV-specific CTL activity was detected in both PBMC and
LNMC of all study subjects and, comparing unfractionated
versus CD25?cell-depleted MC, significant CD25?cell-
mediated suppression of HIV-specific CTL activity was ob-
served with both PBMC and LNMC (Fig. 6A). Of interest, a
difference in the ability of LN CD25? Tregs to suppress
HIV-specific CTL activity was seen between patients with high,
CD25?cell-mediated suppression of HIV-specific CTL activity
in LNMC of the two subjects with the highest VL (VL ?50,000;
patients 4 and 5) was significantly greater than that observed in
LNMC of subjects with VL ?25,000 HIV RNA copies/ml (Fig.
6B). This difference in CD25?Treg activity related to level of
viremia was not significant with PBMC (Fig. 6B). To determine
whether CD25?Treg cells isolated from the PB and LN differed
in regard to their suppressive capability, LN or PB-derived
CD25hi?or CD25?CD4?T cells were added back (at 10%) to
CD25?MC effector populations at the time of initial HIV Gag
peptide stimulation. Both PB and LN-derived CD25hi?, but not
CD25?, CD4?T cells suppressed HIV-specific CTL activity
(Fig. 6C); however, the suppressive effect of LN-derived
CD25hi?CD4?T cells was significantly greater than that ob-
served with PB-derived CD25hi?CD4?T cells (Fig. 6C).
The present study was designed to determine the functional
capability of lymphoid tissue-derived CD25?Treg cells, partic-
ularly those isolated from individuals with advanced HIV dis-
ease, with regard to their suppressive effects on HIV-specific T
cell responses. The answer to this question is critical to deter-
mining whether immunosuppressive CD25?Treg cells represent
a reasonable target for immune-based approaches for enhancing
HIV-specific immune responses and, if so, whether such an
approach is appropriate at all stages of HIV disease. The results
of this study indicate that CD25?Treg cells maintain significant
suppressive activity against HIV-specific cytolytic T cell re-
sponses even in the advanced stage of HIV disease. Further-
more, LN-derived CD25?Treg cells suppressed HIV-specific
responses to a greater degree than did PB-derived CD25?Treg
cells, particularly in those individuals with high viral loads.
Natural CD25hiFoxP3?Treg cells are an important component
of the immune surveillance system designed to control autoimmu-
nity (1); however, their suppressive activity has been shown to also
impact appropriate foreign antigen-specific immune responses. In
this regard, CD25?Treg cell activity has been shown to contribute
to the persistence of certain infections and tumors in vivo (2–8). In
addition, suppressive antigen-specific CD25?Treg cells can be
generated (‘‘induced’’ CD25?Treg cells) from presumably normal
1000 800 600 4002000
1000 800600400 2000
1000 800 600400 2000
1000 800 600400 2000
1000 800600 4002000
plus CD25+CD8– LNMC
Granzyme-B Substrate Cleavage
Target: No Peptide
Target: plus HIV
a read out. HIV Gag pre-stimulated effector unfractionated (Top plots) and
CD25?cell-depleted LNMC, alone or plus 30% CD25?CD8?MC, (Middle and
Bottom Right plots, respectively) were cultured for 1 h with FL-4-labeled
un-pulsed or HIV Gag peptide-pulsed autologous CD25?CD8?target cells in
substrate cleavage high events within the FL-4?target cell gate; analyses are
of FL-4?target cells only. Substrate cleavage in target cells cultured in the
absence of effector cells is shown in the Bottom Left plot.
Representative example of CD25?Treg suppressive capacity assessed
VL < 25,000VL > 50,000
Average Percent Inhibition of
HIV-specific Cytolytic Activity
Average HIV-specific Cytolytic
Percent Suppression of
HIV-specific CTL Activity
p = 0.007 p = 0.016
p = 0.05
p = 0.016
plus CD25–CD4+ T Cells
plus CD25+hiCD4+ T Cells
p = 0.03
p = 0.03
HIV-specific CTL activity. (A) Mean (? SD) HIV-specific CTL activity in unfrac-
tionated and CD25?cell-depleted PBMC and LNMC. (B) Mean (? SD) percent
inhibition of HIV-specific CTL activity mediated by CD25?cells in LNMC and
PBMC of patients stratified by VL (HIV RNA copies per ml). (C) Comparison of
the suppressive effects of LN and PB-derived CD25hi?CD4?T cells on HIV-
(at 10%) at the time of primary stimulation with HIV Gag peptides. CTL assays
were performed 6 days later after exposure to HIV Gag peptide-pulsed target
cells. Data are of mean (?SD) percent suppression of HIV-specific CTL activity
present in control CD25?cell-depleted MC cultured alone.
Comparison of PB and LN CD25?Treg cell-mediated suppression of
Kinter et al.PNAS ?
February 27, 2007 ?
vol. 104 ?
no. 9 ?
CD4?T cells under certain pathogenic/micro environmental con-
been demonstrated to accumulate and expand at tissue sites of
inflammation/antigen expression where cell contact-mediated sup-
replication/antigen expression largely occurs in the lymphoid tissue
(36, 37), the site at which primary immune responses to most
antigens are generated. Therefore, antigen-driven accumulation
and activation of CD25?Treg cells in lymphoid tissue sites could
have a considerable impact not only on effector cell function but
also on the generation of lymphocyte responses to new antigens or
Results from early studies using PB suggested that
CD25?FoxP3?Treg cell function and/or frequency decline with
cells are not necessarily lost over the course of HIV disease but
rather accumulate in the lymphoid tissue has been generated by
in situ phenotypic and mRNA studies (19, 22, 24). High levels of
HIV replication have been associated with the accumulation of
cells with a Treg-like phenotype and/or FoxP3, TGF-? and IDO
mRNA in tonsils (19, 24) and lamina propria (22). In our
phenotypic studies, CD25hiFoxP3?cell frequencies in LNMC
were consistently higher than in PBMC regardless of viral load
or CD4?T cell counts of the study subject. In contrast, the
frequency of CD25?hiTreg cells is reported to be similar in
lymph node and PB of healthy HIV uninfected controls (40). Of
interest, we also observed that FoxP3 intensity (MFI) in the
CD25?CD4?T cell subset of LNMC was significantly higher
than that in PBMC. It has been reported that human CD25?
Treg cells expressing high levels of FoxP3 exhibit more rapid
suppressive activity compared with FoxP3midCD25?Treg cells
(41). With regard to additional surface markers associated with
CD25?Treg cells, CD25hiFoxP3?CD4?T cells exhibited higher
GITR expression in the LN than in PB, which suggests that LN
Treg cells would be more likely to be activated and expand in
response to interaction with dendritic cells that express GITR-L
(42, 43). CD127neg/lo(29) was the most specific surface pheno-
type for CD25hiFoxP3?CD4?T cells in both PB and LN;
however, a greater proportion of CD25hiFoxP3?Treg cells
expressed CD127hi, rather than CD127neg/lo, in the LN com-
pared with the PB. Overall , no surface marker was sufficient to
fully and accurately discriminate between CD25hiFoxP3?Treg
cells and normal CD4?T cells in the LN.
Importantly, our functional studies demonstrate that CD25?
Treg cells isolated from both the PB and LN of all subjects, even
those with advanced disease, retain significant suppressive ac-
tivity as detected by HIV-specific CTL assays. Although the
sample size in the present study was low, the data suggest that
CD25?Treg cell-mediated suppression of HIV-specific CTL
activity is more effective in lymphoid tissue than in PB, partic-
ularly in those patients with moderate/high compared with
undetectable/low viral loads. Whereas in vitro human CD25?
Treg cells have generally been found to suppress CD4?T cell
proliferation via an ill-defined cell contact-dependent, IL-10/
transforming growth factor (TGF)-?-independent mechanism
(44), in vivo CD25?Treg cells are thought to exert immunosup-
Increased CD25?Treg activity in LNMC compared with PBMC
may be due to differences in the frequency of CD25?Treg cell
subsets that suppress by the production or induction of soluble
immunosuppressive factors (47, 48), particularly TGF-?. Al-
though definitive comparative studies of TGF-? production by
PBMC and LNMC in chronically HIV-infected individuals have
not been performed, TGF-? is elevated in the lymphoid tissue
of viremic HIV-infected subjects (19, 22, 24). Of note, Treg-
mediated suppression of antigen-specific CTL activity in certain
murine models has been shown to be TGF-?-dependent (49, 50).
Using proliferation assays as a read-out, we previously re-
ported on a cohort of patients that exhibited a loss of CD25?
Treg suppressive activity against HIV-specific CD4?T cell
responses in the PB with HIV disease progression (17). We
proposed, as have others (19), that ‘‘HIV-specific’’ Treg cells
might migrate out of the blood into the lymph nodes during
against HIV-specific T cell function would be retained in
lymphoid tissue. However, in the present study, CD25?cell-
mediated suppression of HIV-specific CD4?T cell proliferation
was not observed in either PBMC or LNMC isolated from
individuals with more advanced disease, although suppression of
HIV-specific CTL activity was detected in both PBMC and
LNMC of all subjects. The apparent differential sensitivity of
antigen-specific proliferation and cytotoxicity to CD25? Treg-
mediated suppression has not been reported in the context of
HIV infection. It is possible that factors that reduce sensitivity
of normal T cells to CD25?Treg-mediated suppression, such as
TCR signal strength, GITR expression and IL-6, (28, 43, 51–53)}
differ in the two assay systems. Alternatively, different CD25?
Treg subsets (47) or mechanisms (45, 46) may suppress CTL
effector function more efficiently than other T cell functions. In
this regard, there is evidence in murine models indicating that
CD25?Treg-mediated suppression of CD8? effector functions,
but not of T cell proliferation, requires intact TGF-? signaling
(45, 50, 54). Furthermore, it has been suggested that cytolytic
activity may be the most Treg-sensitive T cell function in vivo
(49). The data from the present study would suggest that CTL
activity in humans is also more sensitive to CD25?Treg-
mediated suppression than is proliferation or other effector
functions, at least in the context of progressive HIV disease.
In summary, the present study provides proof of concept that
CD25?Treg cells maintain the ability to suppress HIV-specific
responses, particularly CTL function, throughout the course of
HIV disease. CD25? Treg-mediated suppression of HIV-
specific cytolytic activity in lymphoid tissue, the major site of
HIV replication, may be particularly detrimental to the ability to
control virus replication in vivo. It remains to be established
whether immune-based therapies designed to transiently deplete
CD25?Treg cells in vivo would ultimately be beneficial or
detrimental in the context of HIV infection.
Materials and Methods
Cellular Subset Isolation. Inguinal or axillary LN biopsies and
lymphophereses were obtained after informed consent from 11
HIV-infected individuals under National Institutes of Health
(NIH) approved protocols (NIH 92-I-25, 02-I-0202, and 81-I-
0164). The clinical profiles of study subjects are shown in Table
1. Fresh LNMC and PBMC of individuals 1–5 were used in
functional assays and frozen LNMC and PBMC from subjects
6–11 were used for phenotypic studies only. LNMC were ob-
tained by gentle physical disruption (scalpel microdissection)
and collagenase treatment. PBMC were isolated by centrifuge
density gradient. MCs or CD4?T cells [isolated by using a
negative selection antibody mixture and immunomagnetic beads
(Stem Cell Technologies, Vancouver, BC, Canada)] were re-
tained as unfractionated or separated into CD25?hiand CD25?
subsets. CD25hicells were obtained by using anti-human CD25
PE conjugated Mab (3 ?l/106cells) followed by a short incuba-
tion (3 min) with anti-PE immunomagnetic beads (Miltenyi,
Auburn, CA). For certain assays, the CD25?MC were further
depleted of CD8?cells by using anti-CD8-coupled immunomag-
netic beads (Dynal, Brown Deer, WI) to derive CD8?CD25?
control MC (?85% CD4?T cells). CD25?Treg phenotypic
analyses were performed on total LNMC and PBMC by using
anti-human FoxP3 (intracellular; EBiosciences, San Diego, CA),
CD4, CD25, CD127, CTLA-4 and GITR. All antibodies were
www.pnas.org?cgi?doi?10.1073?pnas.0611423104Kinter et al.
purchased from BD PharMingen (San Jose, CA) unless other- Download full-text
HIV-Specific Lymphocyte Proliferation Assay. Unfractionated (total)
CD25?or CD25?hiCD4?T cells isolated from lymph nodes or the
1640 (Invitrogen, Carlsbad, CA), 10% human AB serum (Hyclone,
Logan, UT) supplemented with 1 mM glutamine, antibiotics, and
Hepes buffer] plus autologous ?-irradiated CD25?PBMC (APC;
1:1). LN-derived cells were cultured in laminin-coated plates (In-
(5 ?g/ml; Protein Sciences, Meriden, CT) or allo-antigens (a mix of
allogeneic, ?-irradiated PBMC from three different donors cul-
tured at a 1:1 ratio with responder populations). At day 6, post-
stimulation cells were exposed to 0.5 ?Ci per well (1 Ci ? 37 GBq)
proliferation (net cpm). Effective Treg-mediated suppression of
proliferation was considered to be present if net cpm values of
unfractionated MC were ?60% of CD25-depleted MC net cpm
values; this cut-off was based on a similar threshold used in a
previous study (17).
HIV-Specific Cytolytic Activity. All assays were conducted in RPMI
medium 1640 plus 10% human AB sera media. ‘‘Effector’’ (E)
unfractionated or CD25?cell-depleted PBMC or LNMC
(LNMC were cultured in laminin-coated plates) received pri-
mary stimulation with autologous CD25?CD8?MC (peptide-
presenting cells: PPC) (1:10 ratio) that were untreated (no
peptide) or were pulsed with a pool of overlapping 15mer HIV
Gag (AIDS Reagent Repository, Rockville, MD). In certain
experiments the CD25?hiand CD25?subsets of CD4?T cells or
CD8?MC were added to CD25?cell-depleted MC (at 10–30%)
before primary stimulation with peptides. Six days later, effector
cells under each condition were harvested, washed, and 5 ? 106
E cells were restimulated with 0.2 ? 106labeled (FL4 channel
detection) unpulsed or HIV Gag peptide-pulsed, autologous
CD8?CD25?target (T) cells (T:E ratio ? 1:25). Effector plus
target cells were cultured for 1 h in the presence of a Granzyme
B substrate that fluoresces in FL-1 when cleaved (GranToxiLux,
OncoImmunin, Gaithersburg, MD). Granzyme B substrate
cleavage was assessed in the FL4?target population as per
manufactures recommendation by using a BD FACSCalibur.
HIV-specific cytolytic activity was determined by subtracting
substrate cleavage values obtained in cultures containing pep-
tide-free target cells from those values obtained in cultures
containing HIV Gag peptide-pulsed target cells.
Statistical Analyses. All statistical analyses were conducted by
using unpaired or paired Student’s t test and Spearman’s (non-
1. Sakaguchi S, Sakaguchi N, Asano M, Itoh M, Toda M (1995) J Immunol
2. Belkaid Y, Piccirillo CA, Mendez S, Shevach EM, Sacks DL (2002) Nature
3. Belkaid Y, Rouse BT (2005) Nat Immunol 6:353–360.
M, Conejo-Garcia JR, Zhang L, Burow M, et al. (2004) Nat Med 10:942–949.
5. Hisaeda H, Maekawa Y, Iwakawa D, Okada H, Himeno K, Kishihara K,
Tsukumo S, Yasutomo K (2004) Nat Med 10:29–30.
6. Kursar M, Bonhagen K, Fensterle J, Kohler A, Hurwitz R, Kamradt T,
Kaufmann SH, Mittrucker HW (2002) J Exp Med 196:1585–1592.
7. Rouse BT, Sarangi PP, Suvas S (2006) Immunol Rev 212:272–286.
8. Sakaguchi S (2003) Nat Immunol 4:10–11.
10. Lawn SD, Butera ST, Folks TM (2001) Clin Microbiol Rev 14:753–777.
11. Miedema F (1992) Immunodefic Rev 3:173–193.
12. Sheppard HW, Ascher MS (1992) Annu Rev Microbiol 46:533–564.
13. Douek DC, Brenchley JM, Betts MR, Ambrozak DR, Hill BJ, Okamoto Y,
Casazza JP, Kuruppu J, Kunstman K, Wolinsky S, et al. (2002) Nature 417:95–98.
14. Popik W, Pitha PM (2000) Virology 276:1–6.
15. Nixon DF, Aandahl EM, Michaelsson J (2005) Microbes Infect 7:1063–1065.
16. Aandahl EM, Michaelsson J, Moretto WJ, Hecht FM, Nixon DF (2004) J Virol
17. Kinter AL, Hennessey M, Bell A, Kern S, Lin Y, Daucher M, Planta M,
McGlaughlin M, Jackson R, Ziegler SF, Fauci AS (2004) J Exp Med 200:331–343.
18. Weiss L, Donkova-Petrini V, Caccavelli L, Balbo M, Carbonneil C, Levy Y
(2004) Blood 104:3249–3256.
Chougnet CA (2005) J Immunol 174:3143–3147.
20. Apoil PA, Puissant B, Roubinet F, Abbal M, Massip P, Blancher A (2005) J
Acquir Immune Defic Syndr 39:381–385.
21. Oswald-Richter K, Grill SM, Shariat N, Leelawong M, Sundrud MS, Haas DW,
Unutmaz D (2004) PLoS Biol 2:E198.
22. Epple H-J, Loddenkemper C, Kunkel D, Tro ¨ger H, Maul J, Moos V, Berg E,
Ullrich R, Schulzke J-D, Stein H, et al. (2006) Blood 108:3072–3078.
23. Estes JD, Li Q, Reynolds MR, Wietgrefe S, Duan L, Schacker T, Picker LJ,
Watkins DI, Lifson JD, Reilly C, et al. (2006) J Infect Dis 193:703–712.
24. Nilsson J, Boasso A, Velilla PA, Zhang R, Vaccari M, Franchini G, Shearer
GM, Andersson J, Chougnet C (2006) Blood 108:3808–3817.
25. Baecher-Allan C, Brown JA, Freeman GJ, Hafler DA (2003) Novartis Found
Symp 252, 67–88; discussion 88–91, 106–114.
26. Fontenot JD, Gavin MA, Rudensky AY (2003) Nat Immunol 4:330–336.
27. Khattri R, Cox T, Yasayko SA, Ramsdell F (2003) Nat Immunol 4:337–
28. Baecher-Allan C, Viglietta V, Hafler DA (2004) Semin Immunol 16:89–98.
29. Seddiki N, Santner-Nanan B, Martinson J, Zaunders J, Sasson S, Landay A,
Solomon M, Selby W, Alexander SI, Nanan R, et al. (2006) J Exp Med
30. Benito JM, Lopez M, Soriano V (2004) AIDS Rev 6:79–88.
31. Blackbourn DJ, Mackewicz CE, Barker E, Hunt TK, Herndier B, Haase AT,
Levy JA (1996) Proc Natl Acad Sci USA 93:13125–13130.
32. Borrow P, Lewicki H, Hahn BH, Shaw GM, Oldstone MB (1994) J Virol
33. Koup RA, Safrit JT, Cao Y, Andrews CA, McLeod G, Borkowsky W, Farthing
C, Ho DD (1994) J Virol 68:4650–4655.
34. Lichterfeld M, Kaufmann DE, Yu XG, Mui SK, Addo MM, Johnston MN,
Cohen D, Robbins GK, Pae E, Alter G, et al. (2004) J Exp Med 200:701–712.
35. Brenchley JM, Ruff LE, Casazza JP, Koup RA, Price DA, Douek DC (2006)
J Virol 80:6801–6809.
36. Pantaleo G, Graziosi C, Butini L, Pizzo PA, Schnittman SM, Kotler DP, Fauci
AS (1991) Proc Natl Acad Sci USA 88:9838–9842.
37. Pantaleo G, Graziosi C, Demarest JF, Cohen OJ, Vaccarezza M, Gantt K,
Muro-Cacho C, Fauci AS (1994) Immunol Rev 140:105–130.
38. Chen W, Jin W, Hardegen N, Lei KJ, Li L, Marinos N, McGrady G, Wahl SM
(2003) J Exp Med 198:1875–1886.
39. Verhasselt V, Vosters O, Beuneu C, Nicaise C, Stordeur P, Goldman M (2004)
Eur J Immunol 34:762–772.
40. Battaglia A, Ferrandina G, Buzzonetti A, Malinconico P, Legge F, Salutari V,
Scambia G, Fattorossi A (2003) Immunology 110:304–312.
41. Baecher-Allan C, Wolf E, Hafler DA (2006) J Immunol 176:4622–4631.
42. Shevach EM, Stephens GL (2006) Nat Rev Immunol 6:613–618.
43. Stephens GL, McHugh RS, Whitters MJ, Young DA, Luxenberg D, Carreno
BM, Collins M, Shevach EM (2004) J Immunol 173:5008–5020.
44. Baecher-Allan C, Brown JA, Freeman GJ, Hafler DA (2001) J Immunol
45. von Boehmer H (2005) Nat Immunol 6:338–344.
46. Shevach EM (2002) Nat Rev Immunol 2:389–400.
47. Stassen M, Fondel S, Bopp T, Richter C, Muller C, Kubach J, Becker C, Knop
J, Enk AH, Schmitt S, et al. (2004) Eur J Immunol 34:1303–1311.
48. Jonuleit H, Schmitt E, Kakirman H, Stassen M, Knop J, Enk AH (2002) J Exp
49. Khazaie K, von Boehmer H (2006) Semin Cancer Biol 16:124–136.
50. Chen ML, Pittet MJ, Gorelik L, Flavell RA, Weissleder R, von Boehmer H,
Khazaie K (2005) Proc Natl Acad Sci USA 102:419–424.
51. Kubo T, Hatton RD, Oliver J, Liu X, Elson CO, Weaver CT (2004) J Immunol
52. Zheng Y, Manzotti CN, Liu M, Burke F, Mead KI, Sansom DM (2004)
J Immunol 172:2778–2784.
53. Powrie F, Maloy KJ (2003) Science 299:1030–1031.
54. Lin CY, Graca L, Cobbold SP, Waldmann H (2002) Nat Immunol 3:1208–1213.
Kinter et al. PNAS ?
February 27, 2007 ?
vol. 104 ?
no. 9 ?