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Spatial Alterations between CD4+ T
Follicular Helper, B, and CD8 + T Cells
during Simian Immunodeficiency Virus
Infection: T/B Cell Homeostasis, Activation,
and Potential Mechanism for Viral Escape
Jung Joo Hong, Praveen K. Amancha, Kenneth Rogers,
Aftab A. Ansari and Francois Villinger
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Material
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http://www.jimmunol.org/content/suppl/2012/03/02/jimmunol.110313
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The Journal of Immunology is published twice each month by
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Copyright © 2012 by The American Association of
Immunologists, Inc. All rights reserved.
Print ISSN: 0022-1767 Online ISSN: 1550-6606.
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J Immunol 2012; 188:3247-3256; Prepublished online 2
March 2012;
doi: 10.4049/jimmunol.1103138
http://www.jimmunol.org/content/188/7/3247
The Journal of Immunology
Spatial Alterations between CD4+ T Follicular Helper, B, and
CD8+ T Cells during Simian Immunodeficiency Virus
Infection: T/B Cell Homeostasis, Activation, and Potential
Mechanism for Viral Escape
Jung Joo Hong,* Praveen K. Amancha,* Kenneth Rogers,* Aftab A. Ansari,† and
Francois Villinger*,†
T
J.J.H., P.K.A., K.R., and F.V. designed the study; J.J.H., P.K.A., and K.R. performed
the experiments; and J.J.H., P.K.A., A.A.A., and F.V. wrote the paper.
to be upregulated primarily upon chronic activation. Ligation of
PD-1 after cross-linking with its cognate ligands PD-L1 or PD-L2
generally delivers inhibitory regulatory signals to T cells (6, 7).
Murine and human GC T cells, compared with T cells from other
lymphoid tissues, express relatively high levels of PD-1 (8, 9).
The GC helper T cells not only serve as helper cells for humoral
immune responses, but also, due to their expression of CD4, these
T follicular helper (TFH) cells may serve as targets for HIV and
SIV and serve as potential viral reservoirs. Several reports have
shown that at these sites, GC CD4+ T cells are exposed to and are
highly susceptible to HIV (10, 11). The follicular dendritic cells
(FDCs) are thought to trap and transfer virus particles to susceptible T cells, leading to HIV replication in GC CD4+ T cells (12,
13). However, the significance and/or potential role of PD-1+ GCassociated CD4+ T cells during the various phases of infection
remains to be elucidated. In this study, we investigated the distribution and concentration of PD-1hi TFH cells in lymph nodes
collected sequentially from rhesus macaques before and after SIV
infection. In addition, the cellular distribution of SIV and alterations in the levels of B cell activation at various stages of infection were also studied. Our findings suggest that GC CD4+
T cells may not only promote B cell activation and maturation but
also serve as a uniquely “protected” reservoir during chronic infection.
Address correspondence and reprint requests to Dr. Francois Villinger, Division of
Pathology, Yerkes National Primate Research Center, Emory University, 954 Gatewood Road, Atlanta, GA 30329. E-mail address: [email protected]
Materials and Methods
he differentiation of B cells into memory B cells and
their affinity maturation and differentiation into long-lived
plasma cells both take place within germinal centers (GCs)
of secondary lymphoid follicles. After the establishment and
maintenance of GC in the presence of activated helper T cells,
these T cells enhance B cell proliferation, isotype switching, affinity maturation, and differentiation into memory B cells (1, 2).
GC-associated CD4+ helper T cells are distinguished from other
CD4+ T cell subsets phenotypically by their expression of readily
detectable levels of CXCR5, ICOS, Bcl-6, and programmed death1 (PD-1) (3) and functionally by their synthesis of high levels of
IL-21 (4). IL-21 is a common g-chain cytokine that is critical for
GC B cell survival and differentiation (5). PD-1 belongs to the
family of CD28 costimulatory receptors that are generally thought
*Division of Pathology, Yerkes National Primate Research Center, Atlanta, GA
30329; and †Department of Pathology & Laboratory Medicine, Emory University
School of Medicine, Atlanta, GA 30322
Received for publication November 2, 2011. Accepted for publication January 31,
2012.
This work was supported by National Institutes of Health Grants R01AI 078775 and
R24016988 (to F.V.) and DRR000165 (to Yerkes National Primate Research Center)
and by the Emory Center for AIDS Research.
The online version of this article contains supplemental material.
Abbreviations used in this article: DC, dendritic cell; FDC, follicular DC; GC,
germinal center; PD-1, programmed death-1; TFH, T follicular helper.
Copyright Ó 2012 by The American Association of Immunologists, Inc. 0022-1767/12/$16.00
www.jimmunol.org/cgi/doi/10.4049/jimmunol.1103138
Animals
Twenty-seven adult Indian rhesus macaques were used in this study. All
animals were maintained at the Yerkes National Primate Research Center of
Emory University in accordance with the regulations of the Committee on
the Care and Use of Laboratory Animal Resources. The experiments were
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HIV/SIV infections induce chronic immune activation with remodeling of lymphoid architecture and hypergammaglobulinemia,
although the mechanisms leading to such symptoms remain to be fully elucidated. Moreover, lymph nodes have been highlighted as
a predilection site for SIV escape in vivo. Following 20 rhesus macaques infected with SIVmac239 as they progress from preinfection to acute and chronic infection, we document for the first time, to our knowledge, the local dynamics of T follicular
helper (TFH) cells and B cells in situ. Progression of SIV infection was accompanied by increased numbers of well-delineated
follicles containing germinal centers (GCs) and TFH cells with a progressive increase in the density of programmed death-1 (PD-1)
expression in lymph nodes. The rise in PD-1+ TFH cells was followed by a substantial accumulation of Ki67+ B cells within GCs.
However, unlike in blood, major increases in the frequency of CD27+ memory B cells were observed in lymph nodes, indicating
increased turnover of these cells, correlated with increases in total and SIV specific Ab levels. Of importance, compared with T cell
zones, GCs seemed to exclude CD8+ T cells while harboring increasing numbers of CD4+ T cells, many of which are positive for
SIVgag, providing an environment particularly beneficial for virus replication and reservoirs. Our data highlight for the first time,
to our knowledge, important spatial interactions of GC cell subsets during SIV infection, the capacity of lymphoid tissues to
maintain stable relative levels of circulating B cell subsets, and a potential mechanism for viral reservoirs within GCs during SIV
infection. The Journal of Immunology, 2012, 188: 3247–3256.
3248
approved by the local institutional animal use and care as well as biosafety
review boards. Twenty animals were inoculated intravenously with 200 TCID50
(50% tissue culture infective dose) SIVmac239 and served as a source of
blood and inguinal lymph nodes at various time points postinfection.
Quantitation of SIV RNA in plasma
Plasma SIV viral load was determined by quantitative RT-PCR by the
National Institutes of Health Center for AIDS Research sponsored Virology
Core Laboratory of Emory University School of Medicine (14).
Plasma levels of IgM, IgG, and SIV-specific Abs
FIGURE 1. Immunohistological profile
of PD-1–, CD3-, CD4-, CD8-, and CD20-expressing cells within lymph node sections
from a representative SIV-naive rhesus macaque. (A) The section was stained with antiCD20 (blue) to localize lymphoid follicles
and (B) stained with anti–PD-1 (green) and
anti-CD3 (red) to highlight PD-1 expression
on CD3- and CD20-expressing cells. (C) The
staining profile of predominant follicles (few
PD-1hi–expressing cells). (D) The staining
profile of a minor number of lymphoid follicles with intense PD-1–expressing CD3+
T cells within the GC. Images in (C) and (D)
are enlarged from the white boxes in (B) and
collected using a 320 objective. Scale bars,
50 mm. (E–G) Enlarged images of boxed area
shown in (D). Consecutive section of the
same lymph node tissue was stained with
anti-CD8 and anti–PD-1. (H) The profile of
CD8 (blue). (I) The profile of PD-1 (green).
(J) The merged images of (H) and (I). Similarly, the next tissue section was stained with
anti-CD4 and anti–PD-1. (K) The profile of
CD4 (blue). (L) The profile of PD-1 (green).
(M) The merged images of (K) and (L). Scale
bar, 50 mm.
Immunofluorescence staining and quantitative image analysis
Inguinal lymph nodes were biopsied from SIV-naive monkeys and during acute
(14 d postinfection) and chronic (112–131 d postinfection) SIV infection and
embedded unfixed in OCT medium. Seven-micrometer-thick sections were
fixed with 4% paraformaldehyde for 10 min, followed by washing in PBS–
0.1% BSA. The sections were blocked (FcR blocker; Innovex Biosciences,
Richmond, CA) in PBS–0.1% BSA–0.1% Triton-X–4% donkey serum for
30 min. Subsequently, the sections were incubated with goat anti-human PD-1
(R&D Systems, Minneapolis, MN), mouse anti-human Ki67 (clone MM1;
Vector, Burlingame, CA), rabbit or mouse anti-human CD20 (Thermo Scientific, Rockford, IL; or clone L26, Novocastra), mouse anti-human CD4 and
CD8 (clone BC/1F6 and LT8; Abcam, Cambridge, MA), and rat anti-human
CD3 (clone CD3-12; AbD Serotec, Oxford, U.K.) Abs diluted 1:20 to 1:100 in
blocking buffer for 1 h. Thereafter, the sections were washed three times with
blocking buffer and incubated with Alexa Fluor 488, Cy3 or Cy5 conjugated
appropriate donkey anti-mouse, rat or goat Abs, respectively, diluted 1:2000
(Jackson ImmunoResearch, West Grove, PA) in blocking buffer for 30 min.
Tissue sections treated in parallel with mouse and goat IgG isotype Abs were
used as controls to confirm the staining specificity. Finally, the sections were
washed and mounted using warmed glycerol gelatin (Sigma) containing
4 mg/ml n-propyl gallate (Fluka, Switzerland). Images were collected with
an Axio Imager Z1 microscope (Zeiss) using various objectives. For quantification, the area stained with CD20 (follicle) was calculated using the AxioVs40 V4.8.1.0 program (Zeiss). Positive fluorescence signal of PD-1, Ki67,
and PD-L1 within follicles was measured using Image J1.43u (National
Institutes of Health). PD-1+CD3+ and CD3+CD8+ double-labeled cells were
manually counted using FluoView software 1.7 (Olympus). The scoring was
performed in a blinded fashion by three independent observers.
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The ELISA and all staining techniques were performed at room temperature. Plasma levels of total IgM and IgG were determined using commercially available ELISA kits (Life Diagnostics). To quantitate SIVspecific Abs, SIVmac239 virions were grown in serum-free media, inactivated by incubating with 1% Triton X-100 for 30 min, and then captured
onto Maxisorp plates previously coated with 2.5 mg ConA/well. After
a washing step, the wells were blocked with buffer containing 4% whey,
5% dry milk, and 0.01% Tween 20 in PBS for 1 h. Serial plasma dilutions
were added to the wells containing the captured virions and incubated for 1
h. After extensive washing, biotin-conjugated goat anti-human IgG or IgM
(Millipore, Billerica, MA) diluted 1:5000 in blocking buffer was added and
incubated for 1 h followed by the addition of 1:1000 dilution of streptavidin–HRP (Southern Biotech, Birmingham, AL) in blocking buffer. The
plate was then incubated for 1 h, washed, and 100 ml TM Blue substrate
(KPL, Gaithersburg, MD) was added to each well, and the reaction was
stopped by the addition of 2 N sulfuric acid and the OD450 recorded in an
automated ELISA plate reader.
ACTIVATION OF FOLLICULAR T AND B CELLS BY SIV
The Journal of Immunology
Immunohistochemistry
In situ histochemistry was performed on 4-mm tissue sections that were
dewaxed and rehydrated with ddH2O as previously described (15). Heatinduced epitope retrieval was performed with DIVA Decloaker and then
blocked with SNIPER Reagent (Biocare, Walnut Creek, CA) for 15 min.
The sections were incubated with an SIVgag mAb (clone FA2; AIDS Reagent Repository Program) (16) and rabbit anti-CD4 and anti-CD8 polyclonal Abs (Abcam) for 1 h, followed by Mach2 Double-stain detection kit
(Biocare Medical) for 30 min. Abs were detected by development of the
chromogens (Vulcan Fast Red and Vina Green Chromogen Kit; Biocare
Medical). Images were collected with the Axio Imager Z1 microscope.
FACS analysis
Statistical methods
All statistical analyses were performed by using GraphPad Prism (version
5.03) and GraphPad instat (version 3.10). For the comparison of two time
points, the Mann–Whitney U test (two-tailed p value) and the Wilcoxon
matched pairs test (two-tailed p value) were used. The level of correlation
was assessed by Spearman’s rank correlation test.
Results
Follicular lymph node CD4+ T cells within GCs show intense
expression of PD-1
To address the normal distribution of PD-1–expressing cells within
lymph nodes, we first performed in situ analyses for PD-1 expression in CD20+ cell-rich areas, termed as follicles (Fig. 1A),
within which GCs form for optimal Ag presentation to B cells in
SIV-naive rhesus macaques. As expected in these animals, few
GCs were seen that contained a modest number of TFH cells.
However, even in SIV-naive monkeys, TFH cells appeared to express relatively high levels of PD-1 compared with low to undetectable expression of PD-1 on T cells in the paracortical areas of
lymph nodes (Fig. 1B–G). Moreover, the PD-1hiCD3+ T cells were
not uniformly distributed and appeared clustered in one area of the
GC in tissues from these animals (Fig. 1B, 1D). Similar staining
patterns were seen in histological sections of the spleen (data not
shown). Most PD-1hi T cells in follicles were positive for CD4, but
not CD8 (Fig. 1H–M), suggesting that they are CD4+ TFH cells
expressing high relative levels of PD-1 (3) even in healthy animals. In fact, CD8+ T cells seem to be essentially excluded from
GCs (Fig. 1H–J).
Marked accumulation of PD-1hi TFH cells within GC during
chronic SIV infection
Chronic immune activation is a hallmark of HIV/SIV infection (18,
19) characterized by increased frequencies of lymphoid follicles
and GC development postinfection. However, the modulation and
distribution of PD-1hi TFH cells has not formally been investigated
in this context. We therefore investigated whether SIV infection
induced alterations of GC-associated immune architecture, as hypergammaglobulinemia and polyclonal B cell activation are a
common occurrence in HIV-1/SIV infection (20). Although a slight
increase in the frequency of PD-1hi TFH cells was observed in
lymph node sections during peak viremia (day 14 postinfection),
the values were not significantly different from those of tissues
from SIV-naive animals. However, during chronic infection (day
133 postinfection), marked differences were noted. Thus, the
number of follicles containing GC and PD-1hi–expressing T cells
was markedly increased in lymph node sections from chronically
infected animals compared with that in healthy and acutely infected
animals (Fig. 2A, 2B), and the number of follicular PD-1hi T cells
positively correlated with the size of lymph node follicles from
acutely and chronically SIV-infected rhesus macaques, respectively
FIGURE 2. Immunohistological evaluation of PD-1hi–expressing CD3+ T cells in lymph node sections of a representative rhesus macaque as a function
of time after SIV infection. (A and B) The staining profile of CD20- (blue, Cy5), CD3- (red, Cy3), and PD-1– (green, Alexa Fluor 488) expressing cells
within lymph node sections from macaques during acute infection (day 14) (A) and during chronic infection (day 133) (B) (montage original magnification
320). White arrowhead indicates follicle containing GC and PD-1hi–expressing T cells in (A) and (B). Lymph node sections from 10 SIV-infected macaques
obtained during acute infection (day 14, blue diamond) and during chronic infection (day 133, red open square) were examined for the frequency of PD-1hi–
expressing T cells based on follicle size (mm2) as seen in (C). The correlation was assessed by Spearman’s rank correlation test. The p values ,0.05 were
considered statistically significant. (D) Comparative analysis of PD-1hi–expressing T cells in seven uninfected (UN) and the same 10 SIV-infected macaques shown in (C). Plasma viral load of the 20 SIV-infected animals used for this study is depicted in (E). Statistical analyses were performed using Mann–
Whitney U test (two-tailed p value) and Wilcoxon matched pairs test (two-tailed p value). A p value ,0.05 was considered statistically significant.
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Inguinal lymph node cells (1 million) isolated as previously described (17)
were incubated with optimized concentrations of anti-CD3–Alexa Fluor
700 (clone SP34-2), anti-CD8–Pacific blue or anti-CD8–allophycocyanin–
Cy7 (clone RPA-T8), anti-CD20–allophycocyanin (clone 2H7), antiCD21–PE–Cy5 (clone B-ly4), anti-CD27–Pacific blue (clone M-T271)
(BD Biosciences), anti-rhesus CXCR5–FITC (clone 710D82.1), anti-CD4–
AmCyan (clone L200) (National Institutes of Health Nonhuman Primate
Reagent Resource), anti–PD-1–PE–Cy7 (clone EH12.2H7), and antiCCR7–allophycocyanin–Cy7 (clone 3D12) (BioLegend). After washing,
cells were fixed with 1% paraformaldehyde. Data were acquired on an
LSRII flow cytometer (BD Biosciences) and the data analyzed using
FlowJo software (version 9.2; Tree Star, Ashland, OR).
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(Fig. 2C). In addition, the frequencies of PD-1+ T cells/mm2 were
significantly higher within lymphoid follicles from chronically
SIV-infected macaques compared with those in acutely infected or
SIV-naive animals (Fig. 2D, p = 0.0059). Of note, most if not all
PD-1hi TFH cells enumerated from the follicles in Fig. 2C and 2D
were indeed positive for CD4 (data not shown). There was no
significant difference in the frequencies of PD-1hi–expressing cells
in sections from lymph nodes of SIV-naive and acutely infected
macaques (p = 0.2065). After i.v. infection, a typical viral load
profile with a peak around week 2 was observed, followed by a
decline to stable viral load set points of .105 copies/ml of plasma
during the chronic phase of infection (except one animal that
showed spontaneous virus control) (Fig. 2E). These data suggest
that PD-1hi TFH cells accumulate during hyperplasia of follicles
induced by continuous SIV replication in the setting of an organized immune response. We also found that most follicular PD-1hi
T cells expressed CD4, whereas only a few follicular PD-1hi T cells
were CD8+ within GCs (data not shown).
Single-cell suspensions of CXCR5 positive/negative subsets of
lymph node cells from macaques prior to and after SIV infection
were analyzed for levels of PD-1 expression by TFH cells in efforts to
confirm the in situ data. Subsets of T cells infiltrating B cell areas of
lymphoid tissues were defined by the expression of CXCR5 (21, 22)
FIGURE 3. Flow cytometric analysis of lymph node
cells from seven SIV-naive (UN) and 20 SIV-infected
rhesus macaques after acute (day 14) and chronic (day
112–133) infection. (A) The gating strategy to identify
the frequency of CD3+, CD4+, CXCR5+ cells that express PD-1 in a representative sample on day 14 and
day 112–133 postinfection. The isotype controls are
also shown. (B and C) The mean frequency of PD-1–
expressing CD3+ (total) T cells and CD3+CD4+ T cells
that are CXCR52 and CXCR5+ in lymph node cells
from seven SIV-naive (UN) and 20 SIV-infected animals after acute (day 14) and chronic (day 112–133)
infection. (D and E) Mean fluorescence intensity (MFI)
values obtained on the same samples. Statistical analyses were performed using Mann–Whitney U test
(two-tailed p value) and Wilcoxon matched pairs test
(two-tailed p value). Horizontal lines indicate medians.
A p value ,0.05 was considered statistically significant.
(Fig. 3A). Of note, none of the CXCR5 reagents currently available
were able to detect macaque CXCR5 in situ, preventing us from
verifying that GC CD4+ T cells all expressed CXCR5. As expected,
whereas the frequency and density of PD-1 expression increased
significantly on all T cells as a function of time after SIV infection
(Fig. 3B–E), the values for PD-1 expression did not markedly differ
between CXCR5+ and CXCR52 subsets of CD4+ T cells in samples
from SIV-naive monkeys (Fig. 3B–E). During acute infection, the
frequency and density of PD-1–expressing cells were already significantly higher in both CXCR5+ and CXCR52 total and CD4+
T cells, suggesting that overall activation of lymph node T cells
had already been initiated (Fig. 3B–E, p , 0.01 for total T cells and
p , 0.02 for CD4+ T cells). Similar differences were observed
in samples collected 3 mo later, during the early chronic phase of
infection. The frequencies and density of PD-1 increased during
chronic infection in both CXCR5+ and CXCR52 total and CD4+
T cells compared with values observed pre-infection and during the
acute infection. In addition, the difference in PD-1 expression on
CXCR5+ T cells and CD4+ T cells versus their CXCR52 counterpart was even more prominent (Fig. 3B–E).
The high frequency of PD-1hi TFH cells within GC correlates
with the generation of CD27+ memory B cells and Ab
production during chronic SIV infection
GC interactions dependent on CD4+ TFH cells contribute to the
establishment of B cell memory and effective humoral immune
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Confirmation of levels of PD-1 expression on TFH cells during
chronic SIV infection by flow cytometry
ACTIVATION OF FOLLICULAR T AND B CELLS BY SIV
The Journal of Immunology
node CD21+/CD272 B cells was readily seen as early as during
acute infection (p = 0.0001), which decreased further during the
chronic phase (p = 0.002). This decrease was compensated for by
significant increases in CD27+ memory B cells while the relative
representation of CD212/CD272 B cells did not change (Fig. 5B).
In particular, the transition from acute to chronic phase was associated with an increase in CD212/CD27+ B cells (p , 0.01), but it
is uncertain whether these cells are equivalent to activated memory
B cells observed in blood or plasma cells/plasmablasts that would
be consistent with the observed hypergammaglobulinemia. Although a better definition of lymphoid B cells in macaques is still
being assessed using new Abs specific for various B cell markers
(CD19, CD38, CD77, etc.), the results have important implications
for the homeostasis of B cells during SIV infection. After SIV
infection, whereas two fast progressor monkeys failed to generate
detectable anti-SIV Abs, the remaining normal progressors developed detectable SIV Ab titers with relatively modest changes in
their frequency of peripheral blood B cells subsets (25) (Fig. 5C).
However, within lymph nodes, marked B cell proliferation occurred with the observed shifts in subset distribution (Fig. 5B),
likely resulting in increased B cell turnover and death as they
emigrate into the peripheral circulation. Thus, in normal progressor
monkeys, there appears to be a balance between the supply of new
B cells and decrease of at least the activated memory B cell subsets. To determine the biological significance of the increase in
memory B cell subsets within lymph nodes, we measured the levels
of total IgM, IgG, and SIV-specific IgG in the plasma of the 20
animals as a function of time postinfection. As expected, the
monkeys demonstrated statistically relevant increases in total
plasma IgG including SIV specific IgG during the chronic stage
(Fig. 5D and Supplemental Fig. 1A), which reflects the hypergammaglobulinemia characteristic of both HIV/SIV infection.
IgM levels showed a non-significant decrease over the same time
period (Fig. 5E). Notably, the frequency of follicular PD-1+ T cells
detected in situ positively correlated with total IgG levels, but not
FIGURE 4. Identification of CD20+ cells that are predominantly Ki67+ in lymph node sections from SIV-infected rhesus macaques during chronic
infection. (A) Lymph node section from a representative chronically infected animal was stained with anti-CD3 (red), anti–PD-1 (green), and anti-CD20
(blue). (B) The next consecutive section of the same lymph node was stained with anti-Ki67, and (C)–(E) represent the profiles seen with anti–PD-1 (C),
anti-CD20 (D), and anti-Ki67 (E). (F) The merged image of (C)–(E). The boxed area in (F) is shown to highlight the expression of Ki67 by CD20+ cells.
Scale bar, 50 mm. The intensity of individual images of single follicles obtained using anti–PD-1 (green) and anti-Ki67 (blue) was measured in lymph node
section of SIV-naive and SIV-infected animals after acute and chronic infection. A correlation between Ki67 and PD-1 expression within follicles was
determined. Data of individual follicles from the SIV-naive (red square), day 14 SIV-infected (green triangle), and day 133 SIV-infected (blue diamond)
animals is shown in (G). The correlation was assessed by Spearman’s rank correlation test. The p values ,0.05 were considered statistically significant.
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responses (21, 23), and although PD-1 has been linked to immune
exhaustion, it is also a marker of T cell activation (7). We investigated the proliferative status of GC T and B cells in situ as
a function of SIV infection. Ki67+ was readily detectable in GCs
but absent from T cell zones within the lymph nodes (Fig. 4A,
4B), suggesting a general but not strict overlay of PD-1 and Ki67+
cells within the follicles. However, the expression of PD-1 seemed
more restricted geographically than that of Ki67, suggesting that
these two molecules are expressed by different cell lineages. The
fact that most Ki67+ cells were also CD20+ (Fig. 4C–F) suggests
that the majority of the Ki67 signal was associated with B cells
in GCs rather than TFH cells. However, there was a strong positive
correlation between PD-1 and Ki67 coexpression within each
follicle from healthy and SIV-infected animals (r = 0.8454, p ,
0.0001), suggesting an association between Ki67+ and PD-1–
expressing cells (Fig. 4G). The frequency of naive and memory
B cell subsets were also evaluated based on the expression of
CD21 and CD27 (24, 25) (Fig. 5A). First, we confirmed that the
relative distribution of B cells in the blood of SIV-naive rhesus
macaques markedly differed from humans, comprising ∼35%
naive (CD21+/CD272), 5% resting memory (CD21+/CD27+), 40%
activated memory (CD212/CD27+), and 20% tissue memory
B cells (CD212/CD272) (Fig. 5C) similar to the values reported
by Titanji et al. (25). These values did not change significantly
during acute infection with slightly elevated levels of activated
memory B cells in blood during chronic infection (Fig. 5C). Although the definitions of B cell subsets used for blood do not
adequately apply to lymphoid tissues (26), using the same markers
to analyze B cells in lymph nodes pre-infection and postinfection
showed dramatic differences. In SIV-naive animals, the relative
frequencies of B cell subsets differed substantially from blood
(Fig. 5B), with CD21+/CD272 B cells accounting for .70% of
lymph node B cells and CD21+/CD27+ B cells accounting for 12
and ∼5% each of CD212/CD27+ and CD212/CD272 B cells.
Upon infection, significant decreases in the frequency of lymph
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ACTIVATION OF FOLLICULAR T AND B CELLS BY SIV
with IgM (Fig. 5F, 5G). A similar but non-significant trend was
seen between the frequency of PD-1+ T cells and SIV-specific Abs
(r = 0.6667, p = 0.0589) (Supplemental Fig. 1B).
GC predominantly exclude CD8+ T cells, providing for
a replication site and a potential reservoir and escape site for
SIV in follicular CD4+ T cells
GCs induced in secondary lymph nodes are thought to be important
sites of HIV/SIV replication, suggesting that activated TFH cells
could serve as viral targets throughout infection. Moreover, a recent publication has highlighted lymph nodes as a primary site for
viral escape (27). As outlined in Fig. 1, whereas CD4+ T cells and
B cells comprise the most abundant cell populations within GC,
CD8+ T cells appear restricted to the T cell zone but largely excluded from the GC in SIV-naive monkeys. The absence of CD8+
T cells within GC was also true in lymph node sections from SIVinfected animals (Fig. 6D–F). Of interest was the finding of
readily detectable SIVgag p27 staining both within GCs and T cell
zones of lymph nodes collected during the chronic infection stages
(Fig. 6A, 6D), as previously reported (28). These data indicate that
recruitment of CD8+ T cells into GCs does not occur or is very
inefficient, which is further supported by the lack of correlation
between frequencies of CD8+ T cells in GC and frequencies of
PD-1+ TFH cells (Fig. 6H), while the concurrent increase in the
frequency of PD-1hi TFH cells generates additional potential targets for the virus to replicate. Thus, whereas CD8+ T cells may
control infected T cells within the T cell zone, the few CD8+
T cells present within GCs may have little impact on the control
of localized infection, allowing for continuous replication, the
maintenance of viral reservoirs, and perhaps also contributing to
viral escape.
SIV infection promotes PD-L1–PD-1 contact within GCs,
potentially negatively regulating TFH cells
Several laboratories including ours have reported that PD-1–PDL1 interaction limits effector functions of not only CD8 but also
CD4+ T cells in vitro and likely in vivo (29–32). We therefore
examined the lymphoid tissue of our animals for the presence of
PD-L1–expressing cells. As illustrated in Fig. 7, lymph node cells
from control SIV-naive monkeys exhibited PD-L1 mostly as single
scattered cells throughout the node and largely outside of GCs.
In contrast, lymph node cells from animals after SIV infection
showed a markedly enhanced expression of PD-L1 that was almost exclusively localized to GCs and often next to PD-1+ T cells
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FIGURE 5. Increased frequencies of CD27+ memory B cells and PD-1–expressing T cells in the lymph
nodes of chronically SIV-infected rhesus macaques
correlates with hypergammaglobulinemia. (A) The
flow cytometric representative profile of CD21- and
CD27-expressing gated population of CD20+ B cells
in lymph node, as described previously (24, 25), from
SIV-infected macaques during acute and chronic infection. In lymph node (B) and blood (C), the frequencies of CD21+/CD272, CD21+/CD27+, CD212/CD27+,
and CD212/CD272 B cells were determined from SIVnaive (UN) and SIV-infected animals during acute (day
14) and chronic (day 112–133) infection. (D and E)
Total serum IgG and IgM levels in nine rhesus macaques prior to (day 0) and after acute (day 14) and
chronic (day 112–113) SIV infection. (F and G) The
correlation between plasma levels of IgG and IgM, respectively, with frequency of PD-1hi follicular T cells.
Statistical analyses were performed using Wilcoxon
matched pairs test (two-tailed p value) and Spearman’s
rank correlation test. The p values ,0.05 were considered statistically significant.
The Journal of Immunology
3253
(31) (Fig. 7B–E). Of note, we found that whereas cells coexpressing PD-1 and CD3 presented primarily a lymphocytic morphology as expected, cells expressing PD-L1 had more of an
interdigitating morphology consistent with FDC (Fig. 7C–E).
Moreover, there was a strong positive correlation between the
magnitude of PD-L1 and PD-1 expression within each follicle
from healthy and SIV-infected animals (r = 0.8651, p , 0.0001)
(Fig. 7F). Recent studies have shown that the PD-1–PD-L1
pathway plays a key role in the suppression of proliferation and
cytokine production by CD4+ and CD8+ T cells during chronic
viral infections including SIV (31, 33). The relationship between
such PD-1–expressing T cells and PD-L1–expressing cells with
dendritic cell (DC)-like appearance on chronic immune activation
and synthesis/release of cytokines remains to be defined. It is of
interest to note that even though PD-L2 can be induced on bone
marrow-derived DCs in vivo (34, 35), the in situ detection of this
ligand was essentially negative in lymph nodes of monkeys tested
before and after infection (data not shown), suggesting that this
ligand may not play a significant role within this compartment.
Discussion
In the current study, we examined the in vivo spatial and temporal
relations of GC-associated TFH cells, their relative expression
of PD-1, and the activation of B cell subsets during early SIV
infection. Although flow cytometry-based investigations provide
invaluable detail as to the subset and potential function of disassociated lymph node cells, these techniques fail to provide an
appreciation for the spatial distribution of these various subsets.
In addition, the relative expression of receptors across cell subsets
may not be readily apparent. Thus, although PD-1 upregulation
was seen on follicular B cells postinfection using flow cytometry,
the density was very modest compared with TFH cells (data not
shown), and therefore such upregulation was eclipsed by the far
brighter expression on TFH cells using immunofluorescence staining. However, this method provides a direct visual comparison of
relative expression of molecules expressed on various cell lineages.
In this regard, the in situ techniques used in the current study have
allowed us to identify three important findings. First, PD-1+ TFH
cells accumulate within follicles during SIV infection. Second, the
presence of PD-1+ TFH cells has relevance to local/systemic humoral immune responses. Third, there is likely insufficient recruitment of CD8+ T cells into follicles, particularly GCs, which
may contribute toward the development of viral reservoirs (27).
Recent findings have advanced our understanding of the role of
PD-1+ TFH cells, located within the GCs, both in mice and humans
(4, 8, 9, 36). These TFH cells also express high levels of CXCR5,
ICOS, and Bcl-6 (3), and their PD-1 expression was selectively
elevated compared with that of T cells in other areas of lymphoid
tissues, suggesting that GC-associated PD-1hiCD4+ T cells form
a distinct population among T cell subsets. These TFH cells are
characterized by the production of IL-21, which is a critical cytokine for GC B cell survival and differentiation (5). To our
knowledge, a systematic evaluation of the changes that may be
induced within GCs of lymph nodes after pathogenic lentivirus
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FIGURE 6. CD8+ T cells appear predominantly within the T cell zone (TZ)
but not GCs in lymph node sections from
chronically SIV-infected animals. Immunohistological profile of p27-expressing
cells (stained with Vina Green) in lymph
node sections from SIV-infected animals
during chronic infection. A representative
profile shows the image at original magnification 320 (A) and at original magnification 360 for CD4+ cells (stained
with Vulcan Fast Red) (B) and p27expressing cells/CD4+ cells (C). Images of
(B) and (C) are enlarged from the black
box in (A). A representative profile of
lymph node sections stained with antiCD8 and anti-p27 SIVgag is shown at
original magnification 310 in (D) and at
original magnification 340 for GC (E)
and T cell zone (F). The mean frequency
of CD3+CD8+ T cells within follicles of
lymph node cells from SIV-naive (UN,
n = 5) and SIV-infected animals after acute
(day 14, n = 7) and chronic (day 133, n =
7) infection is shown in (G). (H) A lack of
correlation between frequency of follicular CD3+CD8+ cells and frequency of
PD-1+ follicular T cells in the same cohort
of SIV-infected animals during acute (day
14, green triangle) and chronic (day 133,
blue diamond) infection.
3254
ACTIVATION OF FOLLICULAR T AND B CELLS BY SIV
infection has not yet been defined. In this report, we have focused
on the study of PD-1 expression by CD4+ TFH cells and the dynamics of GC B cell activation and the effect of SIV infection. It
has been previously suggested that SIV infection may in fact
disrupt the adequate maturation of high-affinity memory B cells
and plasma cells (37) and hence the rationale for the studies
presented in this report. As expected, we identified CD4+ T cells
showing relatively high levels of PD-1 expression within GCs
compared with that in T cells in other areas of lymphoid tissues of
SIV-naive macaques (Fig. 1). However, the extent of the changes
in the expression of PD-1 in TFH cells clearly highlights the
profound changes that occur within the lymphoid environment
after SIV and by extension HIV infection. A significant number
of PD-1hi–expressing follicular T cells accumulate within hyperplastic follicles of SIV-infected macaques during chronic infection, relative to SIV-naive or SIV-infected macaques during acute
infection (Fig. 2). Of note, these changes were not correlated with
viral loads, as PD-1 expression increased further during chronic
infection while viral loads decreased from their peak level. This
lack of correlation may be secondary to the fact that lymph nodes
may not be the major source of virus production compared with,
for example, the gastrointestinal tract (38, 39), a fact that is also
suggested by the rapid destruction of TFH cells in the lymph nodes
of rapid progressors, despite very high viral loads (Supplemental
Fig. 2). We confirmed the enhanced expression of PD-1 by
CXCR5+CD4+ T cells after SIV infection using flow cytometry,
but could not do so in situ, due to the lack of binding reagents
(data not shown). CXCR5 is the chemokine receptor for CXCL13
produced by FDCs (40, 41). When CD4+ T cells encounter DCs
bearing Ag in the T cell zone, they transiently upregulate CXCR5
and downregulate CCR7, resulting in their infiltration into B cell
follicles (42). Although one chemokine alone is not indispensable
for the migration of T cells to lymphoid follicles (8), CXCR5
expression is a specific molecular marker to identify GC-homing
T cells (21, 22). CXCR5+CD4+ T cells showed a higher level of
PD-1 expression compared with that of CXCR52 T cells, but only
after SIV infection (Fig. 3). We also found a progressive increase
in the levels of PD-1 expression by the CXCR5+ T cell subsets as
a function of time after SIV infection. Besides its role as an inhibitor of T cell response, PD-1 also plays an important role in the
regulation of humoral immune responses specifically within GCs as
elegantly reported by the laboratory of Dr. Shlomchik (43). Thus,
mice deficient in PD-1 or its ligands presented markedly lower
numbers of long-lived plasma cells (43). Of interest however was
the finding that the remaining plasma cells in the absence of PD-1
regulation exhibited higher affinity for their Ag. Although it remains to be established whether such findings translate to humans
and primates, the findings nevertheless provide potential insights
into the mechanisms leading to the hypergammaglobulinemia
commonly observed in HIV-1+ patients and SIV-infected monkeys
(20), as not only was PD-1 expression upregulated within GCs but
also PD-L1 on follicular cells with DC morphology (Fig. 7). The
marked upregulation of PD-1 on TFH cells, which in our studies
accompanied the upregulation of Ki67 on B cells colocalized with
PD-1+ TFH cells during chronic infection, may lead to activation
and differentiation of increased numbers of long-lived plasma
cells. We submit that such interaction likely drives the activation
and differentiation of B cells leading to the diverging distribution
of B cell subsets in blood (Fig. 5C) (25) and corresponding lymph
nodes during SIV infection. Unfortunately, a precise phenotype
of plasma cells, plasmablasts, and the various subpopulations of
B cell precursors in lymph nodes of rhesus macaques is still being
delineated. Thus, these populations could not be evaluated and
compared at this time. However, although our studies confirmed
that only minor changes occur in circulating B cell subsets in
clinically stable SIV infection (25), marked changes were noted in
lymph nodes, even using our relatively limited phenotypic definitions. Thus, the marked increase in CD21+/CD27+ B cells at the
acute/chronic stage of infection followed by CD212/CD27+ B cells
at the chronic phase in lymph nodes suggest that the output of these
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FIGURE 7. PD-L1–expressing cells are localized within GCs of lymph nodes interdigitating with CD4+PD-1hi cells. Immunohistological profile of PDL1– (red, Cy3), PD-1– (green, Alexa Fluor 488), and CD3- (blue, Cy5) expressing lymph node cells with a representative section from an SIV-naive animal
(A) and chronically SIV-infected animal (B). F, follicle. The boxed image in (B) was magnified, and profiles of PD-L1 (C), CD3 and PD-1 (D), and merged
image of PD-L1–PD-1–CD3 (E) are shown. Scale bar, 20 mm. A correlation between the intensity of PD-1 and PD-L1 expression by lymph node cells
within follicles of representative specimens obtained prior to (red squares, day 0) and during acute infection (green triangles, day 14) and chronic infection
(blue diamonds, day 133) is shown in (F). The correlation was assessed by Spearman’s rank correlation test. A p value ,0.05 was considered statistically
significant.
The Journal of Immunology
Acknowledgments
We are indebted to the staff and veterinarians of the Yerkes National Primate
Research Center for excellent care of the animals. We thank the Emory Center
for AIDS Research Virology Core and Benton Lawson for viral RNA load
determinations. The study has also benefited from reagents obtained from
the National Institutes of Health AIDS Reagent and Reference Program.
Disclosures
The authors have no financial conflicts of interest.
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