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IMMUNOBIOLOGY
Differential Th17 CD4 T-cell depletion in pathogenic and nonpathogenic
lentiviral infections
*Jason M. Brenchley,1 *Mirko Paiardini,2 Kenneth S. Knox,3,4 Ava I. Asher,1 Barbara Cervasi,2 Tedi E. Asher,1
Phillip Scheinberg,1 David A. Price,1,5 Chadi A. Hage,3,4 Lisa M. Kholi,3 Alexander Khoruts,6 Ian Frank,2 James Else,7
Timothy Schacker,6 *Guido Silvestri,2 and *Daniel C. Douek1
1Human Immunology Section, Vaccine Research Center (VRC), National Institute of Allergy and Infectious Diseases (NIAID), National Institutes of Health (NIH),
Bethesda, MD; 2Department of Pathology and Laboratory of Medicine, University of Pennsylvania, Philadelphia; 3Division of Pulmonary and Critical Care
Medicine, Indiana University, Indianapolis; 4Richard L. Roudebush Veterans Administration Medical Center, Indianapolis, IN; 5Department of Medical
Biochemistry and Immunology, Cardiff School of Medicine, University of Cardiff, Cardiff, United Kingdom; 6Department of Medicine, University of Minnesota,
Minneapolis; and 7Yerkes National Primate Research Center, Emory University, Atlanta, GA
Acute HIV infection is characterized by
massive loss of CD4 T cells from the
gastrointestinal (GI) tract. Th17 cells are
critical in the defense against microbes,
particularly at mucosal surfaces. Here we
analyzed Th17 cells in the blood, GI tract,
and broncheoalveolar lavage of HIVinfected and uninfected humans, and SIVinfected and uninfected sooty mangabeys. We found that (1) human Th17 cells
are specific for extracellular bacterial and
fungal antigens, but not common viral
antigens; (2) Th17 cells are infected by
HIV in vivo, but not preferentially so;
(3) CD4 T cells in blood of HIV-infected
patients are skewed away from a Th17
phenotype toward a Th1 phenotype with
cellular maturation; (4) there is significant
loss of Th17 cells in the GI tract of HIVinfected patients; (5) Th17 cells are not preferentially lost from the broncheoalveolar
lavage of HIV-infected patients; and (6) SIV-
infected sooty mangabeys maintain
healthy frequencies of Th17 cells in the
blood and GI tract. These observations
further elucidate the immunodeficiency
of HIV disease and may provide a mechanistic basis for the mucosal barrier breakdown that characterizes HIV infection.
Finally, these data may help account for
the nonprogressive nature of nonpathogenic SIV infection in sooty mangabeys.
(Blood. 2008;112:2826-2835)
Introduction
The classic paradigm for CD4 T-cell differentiation delineates a
pathway in which memory CD4 T cells are defined based on their
ability to produce either interferon (IFN)–␥ (Th1 cells) or interleukin (IL)–4 (Th2 cells).1 However, the exclusivity of this model has
been challenged with the recent description of a CD4 T-cell subset
(Th17 cells) that produces IL-17 and IL-22, but neither IFN-␥ nor
IL-4, in response to stimulation through the T-cell receptor.2-6 IL-17
and IL-22 function in vivo to promote recruitment of neutrophils to
areas of bacterial infection, to induce proliferation of enterocytes
and production of antibacterial defensins.7-9 Hence, Th17 cells
likely play an important role in antibacterial immunity.10,11 In
addition, IL-17 has been shown to play a central role in mouse
models of autoimmunity.3,12-14 Although the majority of data
regarding Th17 cells are derived from experiments in mice, recent
studies have shown that Th17 cells can be identified in the blood of
humans and characterized phenotypically based on expression
patterns of certain chemokine and cytokine receptors, and appear to
have specificity for bacterial and fungal antigens.15-17
Studies have recently shown that in HIV and pathogenic simian
immunodeficiency virus (SIV) infection microbial products translocate from the lumen of the gastrointestinal (GI) tract, which is both
structurally and immunologically damaged during acute phase of
infection, into the systemic circulation; these microbial products
are bioactive in vivo and contribute to immune activation during
the chronic phase of infection.18-22 However, naturally SIV-infected
sooty mangabeys (SM) that do not progress to AIDS or manifest
Submitted May 23, 2008; accepted July 19, 2008. Prepublished online as
Blood First Edition paper, July 29, 2008; DOI 10.1182/blood-2008-05-159301.
chronic immune activation do not develop microbial translocation.19 Because Th17 cells are thought to play a central role in
antibacterial immunity, particularly at mucosal surfaces, we sought
to examine the frequency, phenotype, functionality, antigen specificity, and in vivo infection frequency of Th17 cells in the
peripheral blood (PB), GI tract, and broncheoalveolar lavage of
HIV-infected and uninfected persons and SIV-infected and
uninfected SMs.
Methods
Subjects
Thirty-two HIV-infected antiretroviral therapy-naive, 4 antiretroviraltreated, and 38 HIV-uninfected healthy subjects were recruited for this
study. Clinical details are shown in Table 1. HIV⫹ patients were
classified as being “early” based on HIV seropositivity for less than
4 months with maintained CD4 T-cell counts of greater than 200 CD4
T cells/␮L, “chronic” based on seropositivity for more than 1 year with
CD4 counts greater than 200 CD4 T cells/␮L, and “AIDS” based on a
CD4 count of less than 200 CD4 T cells/␮L. Viral load was determined
using either the Roche Amplicor Monitor assay or the Roche Ultradirect
assay (Roche Diagnostics, Indianapolis, IN). All subjects gave informed
consent in compliance with the appropriate institutional review board
procedures, and this study was performed in accordance with the
Declaration of Helsinki.
The online version of this article contains a data supplement.
*J.M.B. and M.P. as well as G.S. and D.C.D. contributed equally to this study.
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BLOOD, 1 OCTOBER 2008 䡠 VOLUME 112, NUMBER 7
DIFFERENTIAL Th17 CD4 T-CELL DEPLETION
Table 1. Subject cohort
Subject no.
HIV status
CD4 T cells/␮L PB
pVL
4340
1
Chronic
492
2
Chronic
685
4410
3
AIDS
179
12 300
4
Chronic
444
25 600
5
Chronic
410
36 900
6
Chronic
385
14 700
7
Chronic
750
4400
8
Chronic
286
⬎ 1 000 000
9
AIDS
10
Chronic
92
99 787
647
30 488
11
Chronic
308
5100
12
Chronic
814
23 800
13
Chronic
662
11 844
14
Chronic
296
750 000
15
Chronic
576
14 850
16
AIDS
184
37 900
17
Chronic
273
1650
18
AIDS
81
35 000
19
Chronic
827
1020
20
Chronic
478
174 000
21
AIDS
147
4960
22
Chronic
218
23 000
23
Chronic
416
8100
24
Early
254
11 000
25
Chronic
268
6783
26
Chronic
373
36 377
27
Chronic
449
⬍ 50
28
Chronic
296
⬎ 750 000
29
Chronic
450
7270
30
Chronic
439
72 900
31
Chronic
275
133 920
32
Chronic
411
445 000
33
AIDS
4
389 000
34
AIDS
109
498 150
35
Chronic
372
7870
36
HAART
216
⬍ 50
37
HAART
219
⬍ 50
39
HAART
392
⬍ 50
40-78
HIV-negative
ND
0
ND indicates not determined.
Twelve naturally SIV-infected and 10 uninfected SMs were included in
this cross-sectional analysis. All SMs were housed at the Yerkes National
Primate Research Center and maintained in accordance with National
Institutes of Health (NIH) guidelines. This study was approved by the
Emory University Institutional Animal Care and Usage Committee. In
uninfected animals, negative plasma SIV polymerase chain reaction (PCR)
and negative HIV-2 serology confirmed the absence of SIV infection.
Samples
PB mononuclear cells were prepared from venous blood by density gradient
centrifugation. Intestinal mucosa was acquired by endoscopy and biopsy of
the terminal ileum. Patients received standard colonoscopy preparation
(GoLYTELY; Braintree Laboratories, Braintree, MA) and were sedated
with Versed and fentanyl. A colonoscope was passed through the large
intestine and the ileo-cecal valve. At least 8 to 10 biopsy samples were
obtained with cold forceps using standard techniques. Biopsies were placed
in RPMI media supplemented with 10% heat inactivated fetal calf serum
(R10) and shipped on wet ice overnight for T-cell analysis. GI tract samples
were then dissected into 100-␮L fragments and digested in Isocove media
supplemented with 2 mg/mL of type II collagenase (Sigma-Aldrich, St
Louis, MO) and 1 unit/mL of DNase I (Sigma-Aldrich) for 30 minutes at
37°C. After digestion, samples were passed through a 100-␮m filter and
washed twice with R10. From this procedure, approximately 3 ⫻ 106 live
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lymphocytes are routinely isolated. Broncheoalveolar lavage (BAL) cells
were obtained after anesthetizing the upper airways with 4% lidocaine
nebulizer, and the vocal cords and proximal airways with 1% lidocaine
solution. Bronchoscopy with lavage was performed through a fiberoptic
bronchoscope wedged in a subsegmental bronchus of the right middle and
right lower lobes as previously described.23 A volume of 150 mL normal
saline at room temperature was instilled in 50-mL aliquots into the medial
segment of the right middle lobe and repeated in the anterior segment of the
right lower lobe. Subjects were sedated with stadol and midazolam.
Typically, 300 mL was instilled to obtain a return of 125 to 200 mL BAL
fluid. BAL fluid was filtered through 100-␮m Nylon mesh (Tetko,
Elmsford, NY) to remove debris, and was centrifuged at 400g for
10 minutes.
Rectal biopsies in sooty mangabeys
Current guidelines for experiments with SMs, being a protected species,
allowed us to sample rectal biopsies. Fecal material was removed from the
rectum, and a rectal scope/sigmoidoscope was then placed into the rectum.
Rectal biopsies were obtained with biopsy forceps and placed in RPMI
media supplemented with 10% heat inactivated fetal calf serum (R10) and
shipped on wet ice overnight for T-cell analysis. To separate intraepithelial
lymphocytes from lamina propria lymphocytes, Hanks balanced salt
solution containing 5 nM ethylenediaminetetraacetic acid solution was
added to the mucosal tissue and incubated with gentle shaking for 1 hour.
The supernatant, which contains intraepithelial lymphocytes, was then
collected, whereas the remaining tissue was digested in RPMI media
supplemented with 0.75 mg/mL of collagenese for 2 hours at 37°C to obtain
lamina propria lymphocytes. After digestion, samples were passed through
a 70-␮m filter to remove residual tissue fragments.
Flow cytometric analysis
Eighteen-parameter flow cytometric analysis was performed using a
FACSAria or LSR II flow cytometer (BD Biosciences, San Jose, CA).
Fluorescein isothiocyanate, phycoerythrin (PE), Cy7PE, Cy5.5PE, allophycocyanin (APC), Cy7APC, Texas Red PE, violet amine reactive dye, and
Quantum-dot 705 were used as the fluorophores. At least 300 000 live
lymphocytes were collected for all experiments. The list-mode data files
were analyzed using FlowJo (TreeStar, Ashland, OR). Functional capacity
was determined after Boolean gating, and subsequent analysis was performed using Simplified Presentation of Incredibly Complex Evaluations
(SPICE, version 2.9; Mario Roederer, Vaccine Research Center (VRC),
National Institute of Allergy and Infectious Diseases [NIAID], NIH). All
values used for analyzing proportionate representation of responses were
background-subtracted.
Intracellular cytokine assay
Stimulation was performed on fresh or frozen lymphocytes as described
elsewhere.24 Freshly isolated or freshly thawed lymphocytes were resuspended at 106/mL in R10 supplemented with 1 ␮g/mL anti-CD28 and
anti-CD49d antibodies. Anti-CD3 clone 12F6 was used to stimulate T cells
mitogenically through the T-cell receptor at 10 pg/mL. In some cases,
phorbol myristyl acetate (PMA), 5 ng/mL, and ionomycin, 1 ␮M (SigmaAldrich) were used to stimulate T cells mitogenically for comparative
purposes. Peptides used to stimulate HIV-specific T cells were 15 amino
acids in length, overlapping by 11 amino acids and encompassed HIV-1
Gag, Pol, Nef, and Env proteins (corresponding to the sequence of HXBc2)
and were used at 2 ␮g/mL. Streptokinase (SK; Varidase; Wyeth-Ayerst,
Princeton, NJ), Candida albicans (CA; Greer, Lenoir, NC), tetanus toxoid
(Colorado Serum, Denver, CO), influenza (Novartis, Basel, Switzerland),
Epstein-Barr virus (Virusys, Sykesville, MD), cytomegalovirus (Lonza
Walkersville, Walkersville, MD), and heat-killed Staphylococcus aureus
(SA; Calbiochem, San Diego, CA) were used to stimulate antigen-specific
T cells. All stimulations were preformed in the presence of brefeldin-A
(1 ␮g/mL; Sigma-Aldrich) for 16 hours at 37°C. All cells were surfacestained for phenotypic markers of interest and stained after fixation/
permeabilization for cytokines (intracellular cytokine staining, ICS).
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BRENCHLEY et al
BLOOD, 1 OCTOBER 2008 䡠 VOLUME 112, NUMBER 7
Monoclonal antibodies and T-cell phenotyping
Monoclonal antibodies and dead cell dye used for phenotypic and
functional characterization of T-cell subsets were anti-CD3 Cy7APC,
anti-CD8 Quantum-dot 705, anti–IFN-␥ fluorescein isothiocyanate, antiTNF Cy7PE, anti–IL-2 APC, anti-CCR5 APC, anti-CD95 Cy5PE (BD
Biosciences PharMingen, San Diego, CA), anti-CD45RO Texas Red PE,
anti–IL-23R biotin (R&D Systems, Minneapolis, MN), anti-CD27 Cy5PE,
anti-CD28 ECD (Beckman Coulter, Fullerton, CA), anti–IL-17 PE (eBiosciences, San Diego, CA), violet-fluorescent reactive dye (Invitrogen,
Carlsbad, CA), and anti-CD4 Cy5.5PE (Invitrogen). Cells were initially
gated based on characteristic light scatter properties, followed by positive
staining for CD3 without binding to the dead cell dye, and then for CD4
staining without CD8 staining. Memory CD4 T-cell subsets were gated
based on characteristic expression patterns of CD45RO and CD27 (or
CD28 and CD95 for studies with SMs). As naive T cells produce neither
IL-17 nor IFN-␥, and as antigen-specific T cells are not detectable in the
naive T-cell pool, we report the frequency of cytokine-secreting cells is
expressed as percentages of memory T cells.
Quantitative PCR
Quantification of HIV gag DNA in sorted memory CD4 T cells was
performed by quantitative PCR (qPCR) by the 5⬘ nuclease (TaqMan) assay
with an ABI7700 system (PerkinElmer Life and Analytical Sciences,
Waltham, MA) as previously described.25,26 To quantify cell number in each
reaction, qPCR was performed simultaneously for albumin gene copy
number as previously described.27 Standards were constructed for absolute
quantification of gag and albumin copy number.
Clonotypic analysis of TCR sequences
Antigen-specific CD4 T cells sorted based on production of intracellular
IFN-␥ or IL-17 after antigen-specific stimulation. Clonotypic analysis was
performed as described previously using a DNA-based multiplex PCR.28
Results
Th17 cells are detected in PB
Initially, we sought to examine the functionality and phenotype of
IL-17–producing CD4 T cells in the PB of humans. Using mitogenic anti-CD3 stimulation and polychromatic flow cytometry, we
identified IL-17–producing CD4 T cells within both the CD27⫹
and CD27⫺ memory CD4 T-cell compartments (Figure 1B). Naive
CD4 T cells produced neither IFN-␥ nor IL-17 (Figure 1B).
Moreover, IL-17 production and IFN-␥ production were generally
mutually exclusive as only low frequencies of CD4 T cells
produced both IFN-␥ and IL-17. Finally, the IL-17– and IFN-␥–
producing memory CD4 T cells were functionally heterogeneous
as they could also produce TNF and/or IL-2 (Figure 1C).
Antigen specificity of human Th17 cells
Given that Th17 cells can be detected after polyclonal stimulation
in humans, we next sought to examine the antigen specificity of
Th17 cells in vivo. Previous studies have detected Th17 cells in
human PB that are specific for CA and Mycobacterium tuberculosis
antigens.15 These findings suggested that Th17 cells might play an
important role in defense against pathogens that are normally
encountered at mucosal surfaces. Hence, we examined Th17
responses to several antigens from viruses that are encountered at
mucosal surfaces: HIV, adenovirus, cytomegalovirus, influenza,
and Epstein-Barr virus. Although we were able to detect CD4
T cells specific for each virus based on the production of IFN-␥, we
consistently found no virus-specific CD4 T cells that produced
Figure 1. Th17 cells are detected in PB and do not significantly overlap with Th1
CD4 T cells. PB lymphocytes were stimulated with anti-CD3 overnight in the
presence of brefeldin A. Cells were then stained and analyzed by flow cytometry as
described in “Intracellular cytokine assay.” (A) Cells were gated based on characteristic light scatter properties, followed by positive staining for CD3 without binding to
the dead cell dye, and then for CD4 staining without CD8 staining. Naive and memory
CD4 T-cell subsets were gated based on characteristic expression patterns of
CD45RO and CD27 (or CD28 and CD95 for studies with Sooty mangabeys, as shown
in parentheses). (B) Individual subsets of CD4 T cells were analyzed for production of
either IL-17 or IFN-␥. (C) The IL-17– and IFN-␥–producing memory subsets were
further analyzed for the production of TNF and IL-2.
IL-17 in any HIV-infected or uninfected person (Figure 2A).
However, on stimulation with SK, heat-killed SA, tetanus toxoid,
and CA, we detected IL-17 production by CD4 T cells from 13 of
the patients we studied (Figure 2B,C), although not all patients in
our cohort responded to each viral, bacterial, or fungal antigen. We
also observed that, although some CD4 T cells produced IL-17 in
response to SK, CA, and SA, others produced IFN-␥ and found that
these different cytokine-secreting populations, both responding to
SK, contained different clones as defined by their TCRBV CDR3
region (Figure S1, available on the Blood website; see the Supplemental Materials link at the top of the online article). Taken
together, these observations confirm the specificity of Th17 cells
for bacterial and fungal antigens, show that they are phenotypically
and functionally heterogeneous, and suggest that polarization to a
Th1 or Th17 phenotype might not be dictated solely by the nature
of the antigen.
Preferential loss of Th17 CD4 T cells in the GI tract of
HIV-infected patients
The finding that Th17 cells respond to bacterial antigens and are an
important mediator of antibacterial defenses, coupled with previous data demonstrating a compromised GI structural29-31 and
immunologic7,32-34 barrier in HIV-infected patients, led us to study
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BLOOD, 1 OCTOBER 2008 䡠 VOLUME 112, NUMBER 7
DIFFERENTIAL Th17 CD4 T-CELL DEPLETION
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Figure 2. Th17 cells respond to bacterial and fungal antigens. PB lymphocytes from cohorts of HIV-infected and uninfected persons were stimulated with a variety of
viral (A) or bacterial antigens (B) overnight in the presence of brefeldin A and stained as in “Intracellular cytokine assay.” Production of IL-17 and IFN-␥ by memory CD4 T cells
in response to individual antigen-specific stimulation was measured by flow cytometry. (C) The frequencies of memory CD4 T cells from PB lymphocytes of several HIV⫹ (F)
and HIV⫺ patients (E) producing either IFN-␥ or IL-17 in response to bacterial or fungal antigens are shown.
the Th17 cell subset in the GI tracts of HIV-infected and uninfected
persons (Figure 3). We found that anti-CD3 stimulation of GI tract
T cells from HIV-uninfected persons revealed a significantly higher
frequency of IL-17–producing CD4 T cells compared with PB
(P ⬍ .001, Figure 3A). In stark contrast, we found significantly
lower frequencies of IL-17–producing CD4 T cells in the GI tract
of HIV-infected patients compared with uninfected persons
(P ⫽ .008, Figure 3B). This finding was confirmed in a second
cohort of patients using biopsies combined from the terminal
ileum, colon, and rectum after mitogenic stimulation with PMA
and ionomycin (P ⫽ .02, Figure 3C). Moreover, in 2 HIV-infected
and 2 HIV-uninfected persons, we found no differences between
Th17 cell frequencies between biopsies taken from the large and
small bowel (data not shown), and recent studies in SIV-infected
rhesus macaque found similarly low frequencies of Th17 cells in
large and small bowel.21 Notably, Th17 cells remained at low
frequency, even in the 4 patients treated with antiretroviral therapy.
However, we found no significant difference between the frequencies of IFN-␥–producing GI CD4 T cells from HIV-infected and
uninfected persons regardless of which biopsy site or stimulation
method was used (Figure 3D,E). Hence, even though CD4 T cells
are massively depleted from the GI tract of HIV-infected patients,32-34 the IL-17–producing CD4 T cells appeared to be preferentially affected. Moreover, although there may be differences
regarding the degree of overall CD4 T-cell depletion across the GI
tract,32-37 our data suggest that the preferential depletion of Th17
cells occurs throughout the entire GI tract in HIV-infected patients.
Of note, we found that Th17 cells had the same proliferative
capacity on mitogenic stimulation and were not more susceptible to
activation-induced cell death compared with Th1 cells. We also
found similar expression of BCL2 and Ki67 by Th1 and Th17 cells
(data not shown). To determine whether the Th17 cells within the
GI tract could be targets for HIV infection in vivo, we measured
expression of CCR5 by Th1 and Th17 cells within in the GI tracts
of HIV-infected and uninfected persons (Figure 3F). We found that
the majority of both the GI tract Th1 and Th17 cells expressed
CCR5 in uninfected persons. However, although the remaining Th1
cells in the GI tracts of HIV-infected patients maintained expression of CCR5, significantly fewer of the remaining Th17 cells
expressed CCR5. Hence, there was a preferential loss of the
CCR5⫹ Th17 targets for the virus, suggesting that the virus may
play a direct role in their depletion. Moreover, we found a very
slight trend toward a negative correlation between plasma viral
load and the frequency of Th17 cells in the GI tract of HIV-infected
patients (R ⫽ ⫺0.5, P ⫽ .15, data not shown), and recent studies
using large numbers of SIV-infected rhesus macaques found
significant negative correlations between plasma levels of SIV and
the frequencies of Th17 cells in both the small and large bowel.21
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BLOOD, 1 OCTOBER 2008 䡠 VOLUME 112, NUMBER 7
Figure 3. Th17 cells are preferentially lost from the GI tracts of HIV-infected patients. (A) GI tract and PB lymphocytes from HIV-uninfected persons (E) were stimulated
with anti-CD3 in the presence of brefeldin A and stained as in “Intracellular cytokine assay.” GI tract lymphocytes from cohorts of HIV-infected (F) and uninfected persons (E)
were stimulated with anti-CD3 overnight (B,D) or with PMA and ionomycin (C,E) for 4 hours in the presence of brefeldin A and stained as in Figure 1. Frequencies of GI tract
memory CD4 T cells that produce either IL-17 (A-C) or IFN-␥ (D,E) were then measured by flow cytometry. In panels C and E, red symbols signify chronically HIV-infected
patients on HAART. (F) Expression of CCR5 by GI tract Th1 and Th17 cells from HIV-infected (F) and HIV-uninfected (E) patients. (G) The frequency of CD13⫹
myelomonocytic cells within total GI tract leukocytes was assessed after gating for CD45 expression with exclusion of dead cells in cohorts of HIV-infected (F) and uninfected
persons (E). (H) Expression of IL-23R by memory CD4 T cells in the GI tracts of HIV-infected (F) and HIV-uninfected persons (E).
The numbers of Th17 cells in the GI tract were too low, however, to
allow for their purification and measurement of infection frequency. An additional factor that could explain the preferential loss
of Th17 cells compared with Th1 cells in the context of massive
CD4 T-cell depletion overall is a change in the antigen-presenting
milieu of the GI tract. Indeed, we found significantly lower
frequencies of CD13⫹ myelomonocytic cells (comprising macrophages, dendritic cells, and granulocytes) within the GI tracts of
HIV-infected patients compared with uninfected persons
(Figure 3G). As our initial observations for the preferential loss of
Th17 cells from the GI tracts of HIV-infected patients were based
on functional capacity, this finding could be attributed to lack of
functionality rather than the physical loss of Th17 cells per se.
Therefore, we also measured the frequency of CD4 T cells in the GI
tracts of HIV-infected and uninfected persons that expressed the
IL-23 receptor, a surface protein expressed by Th17 cells.16 We
found significantly lower frequencies of IL-23R⫹ memory CD4
T cells in the GI tracts of HIV-infected patients compared with
HIV-uninfected persons (Figure 3H). Hence, phenotypic and func-
tional analysis demonstrated preferential loss of Th17 cells from
the GI tract in HIV infection.
Maintenance of healthy frequencies of Th17 cells in BAL of
HIV-infected patients
We have previously shown that, contrary to events in the
HIV-infected GI tract, there appears to be a sparing of CD4
T cells from depletion in the BAL of HIV-infected patients.38 We
therefore investigated whether the preferential loss of Th17 cells
compared with Th1 cells from the GI tract was also reflected in
BAL. Initially, we compared the frequencies of Th17 cells in
blood, GI tract, and BAL of HIV-uninfected persons (Figure 4A)
and found that the GI tract had significantly higher frequencies
of Th17 cells compared with BAL (P ⫽ .021, Figure 4A).
However, in HIV-infected patients, BAL had higher frequencies
of Th17 cells compared with GI tract and PB (Figure 4C,
P ⫽ .065). Moreover, BAL had higher frequencies of Th1 cells
compared with PB or GI tract in HIV-infected and uninfected
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Figure 4. Th17 cells are not preferentially lost from
the BAL of HIV-infected patients. (A-F) GI tract, PB,
and BAL lymphocytes from HIV-uninfected persons (E)
and HIV-infected patients (F) were stimulated with antiCD3 in the presence of brefeldin A and stained as in
“Intracellular cytokine assay.” Frequencies of memory
CD4 T cells that produce either IL-17 (A,C,E) or IFN␥ (B,D,F) were then measured by flow cytometry. (G) BAL
lymphocytes from 4 HIV-infected patients were stimulated with CA antigen in the presence of brefeldin A and
stained as in “Intracellular cytokine assay.” Production of
either IL-17 or IFN-␥ by memory CD4 T cells is shown.
persons (Figure 4B,D). Thus, different mucosal sites appear to
be affected differentially in HIV infection not only in terms of
the quantity of CD4 T-cell depletion but also in terms of the
functional qualities of the CD4 T-cell subsets therein as
HIV-infected patients maintained healthy frequencies of Th1
and Th17 cells in BAL compared with HIV-uninfected persons
(Figure 4E,F). Importantly, HIV-infected patients generally do
not die of opportunistic infections during the asymptomatic
chronic phase of the infection, and we found that some of the
preserved Th17 cells in BAL responded to CA (Figure 4G).
Th1 skewing on CD4 T-cell maturation in PB of HIV-infected
patients
We next compared the frequency and phenotypes of IL-17–producing
CD4 T cells in PB of HIV-infected and uninfected persons after
anti-CD3 stimulation. We specifically examined the CD27⫹ and CD27⫺
memory CD4 T cells as these memory subsets are maturationally
distinct and the more mature, CD27⫺, subset has been shown to be
expanded in HIV-infected patients.25,39,40 There is considerable phenotypic and functional overlap between the CCR7⫹“central memory”
subset and CD27⫹ memory CD4 T cells and between the
CCR7⫺“effector memory” subset and CD27⫺ memory CD4 T cells. In
doing so, we found no significant differences in the overall frequency of
IL-17-producing CD4 T cells within either the CD27⫹ (Figure 5A) or
CD27⫺ (Figure 5B) memory T-cell subsets in HIV-infected patients
compared with uninfected persons, indicating that their preferential loss
is particular to the GI tract.
However, analysis of the relative frequencies of Th17 cells
compared with Th1 cells among individual memory CD4 T-cell
compartments revealed differences between HIV-infected and
uninfected persons (Figure 5C,D). In HIV-uninfected persons, we
observed significantly higher frequencies of Th17 cells compared
with Th1 cells in the CD27⫺ memory CD4 T-cell subset compared
with the CD27⫹ memory CD4 T-cell subset (P ⫽ .008, Figure 5C).
In contrast, CD27⫹ memory CD4 T cells in HIV-infected patients
were more significantly likely to produce IL-17 compared with
CD27⫺ memory CD4 T cells (P ⫽ .008, Figure 5D). Thus, PB
CD4 T cells from HIV-infected patients were skewed toward Th1
differentiation with maturation toward a CD27⫺ memory phenotype. Together, these data for PB and the GI tract could suggest that
either the proinflammatory state of the immune system in HIVinfected patients leads specifically to a Th1 phenotype or that Th17
cells are preferential targets for the virus in vivo.
Th17 CD4 T cells are not preferentially infected by HIV
in vivo in PB
Thus, to determine whether Th17 cells were preferentially infected
by the virus in vivo, we stimulated PB cells with anti-CD3 and
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BLOOD, 1 OCTOBER 2008 䡠 VOLUME 112, NUMBER 7
BRENCHLEY et al
Figure 5. CD4 T cells in PB of HIV-infected patients
are skewed toward a Th1 phenotype. PB lymphocytes from cohorts of HIV-infected (F) and uninfected
persons (E) were stimulated with anti-CD3 overnight in
the presence of brefeldin A and stained as in Figure 1.
The frequency of memory CD27⫹ (A) and CD27⫺ (B)
CD4 T cells that produced IL-17 or IFN-␥ was then
measured by flow cytometry. The ratio of IL-17–
producing cells to IFN-␥–producing cells was then
compared within both memory CD27⫺ and CD27⫹ CD4
T-cell subsets from HIV⫺ (C, E) and HIV⫹ (D, E)
patients. Statistical significance was determined by the
Mann-Whitney test.
sorted CD27⫹ memory CD4 T cells that produced IL-17, IFN-␥, or
neither by flow cytometry. Their infection frequency was then
determined by qPCR for viral DNA (Figure 6). We specifically
sorted functionally distinct CD4 T cells from only the CD27⫹
memory subset because phenotypic maturation is itself associated
with lower frequencies of infected cells in vivo.25,41 Hence, any
differences between infection frequencies could be directly attributed to functionality rather than maturational phenotype. We found
no significant differences between the infection frequencies of any
individual subset of CD27⫹ memory CD4 T cells based on
production of IFN-␥ or IL-17. Thus, although Th17 cells are
capable of being infected by HIV in vivo and our data suggest that
the virus may play a direct role in their depletion in the GI tract,
Th17 cells in PB do not appear to be preferentially infected.
Th17 cells maintain polyfunctionality with maturation
Use of polychromatic flow cytometry allowed us to examine the overall
functionality of PB Th1 and Th17 CD4 T cells in HIV-infected and
uninfected persons by simultaneous measurement of IL-17, IFN-␥,
TNF, and IL-2 (Figure 7). In doing so, we found that, among both
HIV-uninfected (21 patients) and infected patients (17 patients), CD27⫹
memory CD4 T cells that produce IL-17 were significantly more
polyfunctional than IFN-␥–producing CD27⫹ memory CD4 T cells
(Figure 7A,B). However, within the CD27⫺ memory CD4 T-cell subset
Figure 6. Infection frequencies of Th17 cells in PB. PB lymphocytes from a cohort
of HIV-infected patients were stimulated with anti-CD3 overnight in the presence of
brefeldin A and stained as in “Intracellular cytokine assay.” CD27⫹ memory CD4
T cells that produced IFN-␥, IL-17, or neither were then sorted by flow cytometry, and
the infection frequency was determined by quantitative PCR for viral DNA as
described in “Quantitative PCR.”
of HIV-uninfected persons, functionality of IL-17– and IFN-␥–
producing CD4 T cells was similar (Figure 7C). Analysis of the CD27⫺
memory CD4 T-cell subset from HIV-infected patients revealed that,
unlike the same memory subset within HIV-uninfected persons, the
IFN-␥–producing CD4 T cells were significantly less polyfunctional
than the IL-17–producing subset. Our data thus show a specific
functional deficit in circulating Th1 but not Th17 cells in HIV-infected
patients; this phenomenon may contribute to the overall state of
immunodeficiency. Indeed, recent data have suggested that decreased
functionality of HIV-specific T cells is associated with progressive HIV
disease.42
Th17 cells are maintained at healthy frequencies in the GI tract
in nonpathogenic infection of SMs
SIV-infected SMs do not develop chronic immune activation or
progress to AIDS43 and do not manifest microbial translocation.19
However, such nonpathogenic SIV infection nevertheless results in
overall GI tract CD4 T-cell depletion during acute infection to
levels that are 5% to 20% those of uninfected animals.44 Thus, we
determined whether the finding of decreased Th17 cells is a feature
particular to progressive, pathogenic lentiviral infection. Initially,
we examined expression patterns of IL-17, IL-2, TNF, and IFN-␥
by PB CD4 T cells from SMs and found the Th17 cells in SMs, as
in humans, were functionally heterogeneous and were virtually
mutually exclusive with Th1 cells (data not shown). We then
studied Th1 and Th17 cells in the GI tracts of SMs and found that
SIV-infected SMs maintained frequencies of functional GI tract
Th17 and Th1 cells comparable with uninfected SMs (Figure 8A,B).
In addition, we found similar frequencies of Th17 cell defined
phenopytically (based on coexpression of CD4 and IL-23R) in the
GI tracts of SIV-infected and uninfected SMs (Figure 8C). In PB,
SIV-infected SMs had lower frequencies of Th17 cells compared
with uninfected SMs (P ⫽ .038, Figure 8D). Thus in a nonpathogenic lentiviral infection, even in the context of overall CD4 T-cell
depletion, Th17 cells are not preferentially lost from the GI tract.
Discussion
We examined the frequency, phenotype, functionality, and anatomic distribution of Th17 cells in HIV-infected and uninfected
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BLOOD, 1 OCTOBER 2008 䡠 VOLUME 112, NUMBER 7
DIFFERENTIAL Th17 CD4 T-CELL DEPLETION
2833
Figure 7. Th1 CD4 T cells are less functional than
Th17 CD4 T cells in HIV-infected patients. PB lymphocytes from cohorts of HIV-uninfected and -infected patients were stimulated with anti-CD3 overnight in the
presence of brefeldin A and stained as in “Intracellular
cytokine assay.” The ability of IL-17–producing or IFN-␥–
producing CD27⫹ (A,B) and CD27⫺ (C,D) memory CD4
T cells to coproduce IL-2 and TNF was analyzed using
SPICE as described in “Flow cytometric analysis.” Statistical significance was determined by the Mann-Whitney
test.
humans and in SIV-infected and uninfected SMs monkeys. The
principle findings were as follows: (1) the GI tract is enriched in
Th17 cells compared with PB in healthy patients; (2) CD4 T cells
in blood of HIV-infected patients are skewed away from a Th17
phenotype toward a monofunctional Th1 phenotype with cellular
maturation; (3) PB Th17 cells are infected by HIV in vivo, but not
preferentially so compared with Th1 cells; (4) Th17 cells are
preferentially diminished compared with Th1 cells in the GI tracts
of HIV-infected patients; (5) Th17 cells are preserved in the BAL
of HIV-infected patients; and (6) in nonpathogenic SIV infection of
SMs, healthy frequencies of Th17 cells are maintained in the GI
tract and blood.
Figure 8. Th17 cells are not preferentially lost from the GI tracts of SIV-infected
SMs. GI tract lymphocytes from cohorts of SIV-infected (F) and uninfected sooty
mangabeys (E) were stimulated with PMA and ionomycin for 4 hours in the presence
of brefeldin A and stained as in “Intracellular cytokine assay.” Frequencies of GI tract
memory CD4 T cells that produce either IL-17 (A) or IFN-␥ (B) were then measured
by flow cytometry. (C) Expression of IL-23R by memory CD4 T cells in the GI tracts of
SIV-infected (F) and SIV-uninfected sooty mangabeys (E). (D) Frequencies of PB
memory T cells that produce IL-17 from SIV-infected (F) and uninfected (E) sooty
mangabeys.
Th17 cells play a critical role in antimicrobial immunity by
induction of antibacterial defensins and recruitment of neutrophils.7-9 Moreover, Th17 cells may play a role in GI enterocyte
homeostasis.8,9 Chronic HIV infection is characterized by systemic
depletion of CD4 T cells45 that begins with the massive depletion of
gastrointestinal CD4 T cells during the acute phase of infection,32-34 increased intestinal permeability and enteropathy,46 and
chronic activation of the immune system, which is a significant
predictor of disease progression.47-51 One consequence of the
adverse events that occur in the GI tract during HIV infection is
translocation of immunostimulatory microbial products, which
then directly stimulate the immune system in vivo.18,19 The
preferential and sustained loss of Th17 cells from the HIV-infected
GI tract could therefore have a deleterious effect on the maintenance of the mucosal barrier, both structurally and immunologically, with reduced control of mucosal microbes ensuing and
rendering the patient particularly susceptible to microbial translocation. Indeed, a recent study has shown that CD4 T cells from
patients with dominant negative stat3 gene mutations are unable to
differentiate into Th17 cells in vivo, which renders these patients
exquisitely susceptible to bacterial infections.52
Although Th17 cells are not preferentially infected by HIV
compared with Th1 cells in PB, the specific loss of CCR5⫹ Th17
cells in the GI tract supports a direct role for the virus in their
preferential loss of Th17 cells from the GI tract. In addition, we
propose that the low frequencies of APC we observed within the GI
tracts of HIV-infected patients may contribute to an altered
cytokine environment that would favor the differentiation of CD4
T cells along a Th1 rather than a Th17 pathway.53
Consistent with the supposition that preferential loss of Th17
cells is an important aspect of pathogenic lentiviral infection, Th17
cells are similarly lost from effector tissues in SIV-infected rhesus
macaques.21 However, SMs, which become similarly depleted of
GI tract CD4 T cells during the acute phase of SIV infection but do
not progress to AIDS, do not manifest preferential loss of Th17
cells. These animals are therefore presumably able to maintain
Th17 cell frequencies above a certain threshold, such that the
structural and immunologic integrity of the GI tract remains intact.
Hence, preferential loss of GI tract Th17 cells appears to be a
hallmark of progressive HIV/SIV infection. Although the depletion
of both Th1 and Th17 subsets is probably paramount in the
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BLOOD, 1 OCTOBER 2008 䡠 VOLUME 112, NUMBER 7
BRENCHLEY et al
pathogenesis of HIV infection, the particular depletion of Th17
cells at the mucosal surface, by virtue of their antigen specificity,
may be the more critical in determining bacterial control and
microbial translocation, and thus the pace of HIV disease progression. The mechanisms that underlie such Th17 cell loss in the GI
tract, and the relationship of this phenomenon to GI tract integrity
and microbial translocation, might be central to the development of
therapeutic interventions that modify the consequences of damage
to the GI tract and, by extension, disease progression.
Acknowledgments
The authors thank Beth Zwickl, Mitchell Goldman, and Jeff Waltz
for patient referral, and the Indiana University General Clinical
Research Center for patient care.
This work was supported by NIH grants R01 AI052755 and
AI066998, the Yerkes Primate Center (RR-00165), and the W.W.
Smith Charitable Trust (G.S.); NIH grants R01 AI54232, K24
AI056986, and R01 DE-12934 (T.W.S.); and NIH grants
K08HL04545-05 and R01 HL083468 (K.S.K.). D.A.P. is a Medical
Research Council (UK) Senior Clinical Fellow. This work was
supported in part by the intramural program of NIAID, NIH.
Authorship
Contribution: J.M.B., M.P., A.I.A., B.C., T.E.A., P.S., and D.A.P.
performed the experiments; J.M.B., M.P., G.S., and D.C.D. analyzed the data and conceived the study; and K.S.K., C.A.H.,
L.M.K., A.K., I.F., J.E., and T.S. procured patient and/or animal
samples and recruited and cared for study subjects.
Conflict-of-interest disclosure: The authors declare no competing financial interests.
Correspondence: Jason M. Brenchley, Human Immunology
Section, VRC, Room 301, Building 4, 9000 Rockville Pike,
Bethesda, MD 20892; e-mail: [email protected]; or Daniel C.
Douek, Human Immunology Section, VRC, Room 3509, Building
40, 9000 Rockville Pike, Bethesda, MD 20892; e-mail: ddouek@
nih.gov.
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2008 112: 2826-2835
doi:10.1182/blood-2008-05-159301 originally published
online July 29, 2008
Differential Th17 CD4 T-cell depletion in pathogenic and nonpathogenic
lentiviral infections
Jason M. Brenchley, Mirko Paiardini, Kenneth S. Knox, Ava I. Asher, Barbara Cervasi, Tedi E. Asher,
Phillip Scheinberg, David A. Price, Chadi A. Hage, Lisa M. Kholi, Alexander Khoruts, Ian Frank,
James Else, Timothy Schacker, Guido Silvestri and Daniel C. Douek
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