The PD-1/PD-L costimulatory pathway critically affects host resistance to the pathogenic fungus Histoplasma capsulatum.
ABSTRACT The PD-1 costimulatory receptor inhibits T cell receptor signaling upon interacting with its ligands PD-L1 and PD-L2. The PD-1/PD-L pathway is critical in maintaining self-tolerance. In this study, we examined the role of PD-1 in a mouse model of acute infection with Histoplasma capsulatum, a major human pathogenic fungus. In a lethal model of histoplasmosis, all PD-1-deficient mice survived infection, whereas the wild-type mice died with disseminated disease. PD-L expression on macrophages and splenocytes was up-regulated during infection, and macrophages from infected mice inhibited in vitro T cell activation. Of interest, antibody blocking of PD-1 significantly increased survival of lethally infected wild-type mice. Thus, our studies extend the role of the PD-1/PD-L pathway in regulating antimicrobial immunity to fungal pathogens. The results show that the PD-1/PD-L pathway has a key role in the regulation of antifungal immunity, and suggest that manipulation of this pathway represents a strategy of immunotherapy for histoplasmosis.
- SourceAvailable from: PubMed Central[Show abstract] [Hide abstract]
ABSTRACT: A major pathophysiologic mechanism in sepsis is impaired host immunity which results in failure to eradicate invading pathogens and increased susceptibility to secondary infections. Although many immunosuppressive mechanisms exist, increased expression of the inhibitory receptor programmed cell death 1 (PD-1) and its ligand (PD-L1) are thought to play key roles. The newly recognized phenomenon of T cell exhaustion is mediated in part by PD-1 effects on T cells. This study tested the ability of anti-PD-1 and anti-PD-L1 antibodies to prevent apoptosis and improve lymphocyte function in septic patients. Blood was obtained from 43 septic and 15 non-septic critically-ill patients. Effects of anti-PD-1, anti-PD-L1, or isotype-control antibody on lymphocyte apoptosis and interferon gamma (IFN-gamma) and interleukin-2 (IL-2) production were quantitated by flow cytometry. Lymphocytes from septic patients produced decreased IFN-gamma and IL-2 and had increased CD8 T cell expression of PD-1 and decreased PD-L1 expression compared to non-septic patients (P < 0.05). Monocytes from septic patients had increased PD-L1 and decreased HLA-DR expression compared to non-septic patients (P < 0.01). CD8 T cell expression of PD-1 increased over time in ICU as PD-L1, IFN-gamma, and IL2 decreased. In addition, donors with the highest CD8 PD-1 expression together with the lowest CD8 PD-L1 expression also had lower levels of HLA-DR expression in monocytes, and an increased rate of secondary infections, suggestive of a more immune exhausted phenotype. Treatment of cells from septic patients with anti-PD-1 or anti-PD-L1 antibody decreased apoptosis and increased IFN-gamma and IL-2 production in septic patients; (P < 0.01). The percentage of CD4 T cells that were PD-1 positive correlated with the degree of cellular apoptosis (P < 0.01). In vitro blockade of the PD-1:PD-L1 pathway decreases apoptosis and improves immune cell function in septic patients. The current results together with multiple positive studies of anti-PD-1 and anti-PD-L1 in animal models of bacterial and fungal infections and the relative safety profile of anti-PD-1/anti-PD-L1 in human oncology trials to date strongly support the initiation of clinical trials testing these antibodies in sepsis, a disorder with a high mortality.Critical care (London, England) 01/2014; 18(1):R3. · 5.04 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: Sepsis remains the leading cause of death in most intensive care units. Advances in understanding the immune response to sepsis provide the opportunity to develop more effective therapies. The immune response in sepsis can be characterized by a cytokine-mediated hyper-inflammatory phase, which most patients survive, and a subsequent immune-suppressive phase. Patients fail to eradicate invading pathogens and are susceptible to opportunistic organisms in the hypo-inflammatory phase. Many mechanisms are responsible for sepsis-induced immuno-suppression, including apoptotic depletion of immune cells, increased T regulatory and myeloid-derived suppressor cells, and cellular exhaustion. Currently in clinical trial for sepsis are granulocyte macrophage colony stimulating factor and interferon gamma, immune-therapeutic agents that boost patient immunity. Immuno-adjuvants with promise in clinically-relevant animal models of sepsis include anti-programmed cell death-1 and interleukin-7. The future of immune-therapy in sepsis will necessitate identification of the immunologic phase using clinical and laboratory parameters as well as biomarkers of innate and adaptive immunity.Virulence 09/2013; 5(1). · 3.32 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: Programmed cell death 1 (PD-1) is an inhibitory immune receptor that regulates T cell function, yet the molecular events that control its expression are largely unknown. We show here that B lymphocyte-induced maturation protein 1 (Blimp-1)-deficient CD8 T cells fail to repress PD-1 during the early stages of CD8 T cell differentiation after acute infection with lymphocytic choriomeningitis virus (LCMV) strain Armstrong. Blimp-1 represses PD-1 through a feed-forward repressive circuit by regulating PD-1 directly and by repressing NFATc1 expression, an activator of PD-1 expression. Blimp-1 binding induces a repressive chromatin structure at the PD-1 locus, leading to the eviction of NFATc1 from its site. These data place Blimp-1 at an important phase of the CD8 T cell effector response and provide a molecular mechanism for its repression of PD-1.Journal of Experimental Medicine 03/2014; · 13.91 Impact Factor
The PD-1/PD-L costimulatory pathway critically
affects host resistance to the pathogenic
fungus Histoplasma capsulatum
Eszter La ´za ´r-Molna ´r*, Attila Ga ´cser†, Gordon J. Freeman‡, Steven C. Almo§¶, Stanley G. Nathenson*?**,
and Joshua D. Nosanchuk*†**
Departments of *Microbiology and Immunology,?Cell Biology,†Medicine,§Biochemistry, and¶Physiology and Biophysics, Albert Einstein College of
Medicine, Yeshiva University, 1300 Morris Park Avenue, Bronx, NY 10461; and‡Department of Medical Oncology, Dana–Farber Cancer Institute,
Department of Medicine, Harvard Medical School, 44 Binney Street, Boston, MA 02115
Contributed by Stanley G. Nathenson, December 20, 2007 (sent for review November 30, 2007)
The PD-1 costimulatory receptor inhibits T cell receptor signaling
upon interacting with its ligands PD-L1 and PD-L2. The PD-1/PD-L
pathway is critical in maintaining self-tolerance. In this study, we
Histoplasma capsulatum, a major human pathogenic fungus. In a
lethal model of histoplasmosis, all PD-1-deficient mice survived
infection, whereas the wild-type mice died with disseminated
disease. PD-L expression on macrophages and splenocytes was
up-regulated during infection, and macrophages from infected
mice inhibited in vitro T cell activation. Of interest, antibody
blocking of PD-1 significantly increased survival of lethally infected
wild-type mice. Thus, our studies extend the role of the PD-1/PD-L
pathway in regulating antimicrobial immunity to fungal patho-
gens. The results show that the PD-1/PD-L pathway has a key role
in the regulation of antifungal immunity, and suggest that manip-
ulation of this pathway represents a strategy of immunotherapy
costimulation ? fungal infection ? programmed death-1
?500,000 individuals each year (1). Infection typically results in a
mild, often asymptomatic respiratory illness but may progress to
life-threatening systemic disease, particularly in immunocompro-
mised individuals. Upon inhalation, Hc is ingested by resident
pulmonary macrophages, where the fungus replicates and subse-
quently disseminates to other organs. Macrophages are considered
the most important effector cells in host resistance against his-
toplasmosis by functioning in both innate and cell-mediated im-
munity (2). However, resolution of histoplasmosis depends on the
activation of cell-mediated immunity, in particular effective T cell
responses (1). Both CD4?and CD8?T cells contribute to host
resistance in primary Hc infection. Reduction of CD4?T cells
CD8?T cells, clearance of Hc from organs is impaired (3, 4).
Sublethal infection with Hc evokes a Th1-like response in mice,
the acute phase of infection (5). Upon induction of cell-mediated
immunity and the production of cytokines, macrophages are acti-
vated, and the fungus is eliminated. The importance of B cells in
primary histoplasmosis is less critical (3), however, in B cell-
deficient animals the progression toward lethal infection is accel-
erated in reactivation disease (6).
Programmed cell death-1 (PD-1, CD279) is an immune inhibi-
tory receptor belonging to the CD28:B7 family of costimulatory
molecules, which is expressed on activated T cells, B cells, and
myeloid cells (7). PD-1 binds to two ligands, PD-L1 (B7-H1,
CD274) and PD-L2 (B7-DC, CD273). PD-L2 has higher affinity to
istoplasmosis, caused by Histoplasma capsulatum (HC) is the
most prevalent fungal respiratory disease in the US, affecting
and a variety of nonhematopoietic cell types (8–10). Engagement
of PD-1 by its ligands simultaneously with TCR or BCR cross-
linking induces negative signaling by recruitment of phosphatases
such as SHP-2 and dephosphorylation of effector molecules in-
volved in downstream TCR or BCR signaling (11). PD-1 has a
crucial role in initiating and maintaining peripheral tolerance,
consistent with the finding that PD-1-deficient mice (Pdcd1?/?)
develop various spontaneous autoimmune diseases depending on
the genetic background (12).
Mounting evidence suggests that the PD-1–PD-L pathway plays
a central role in the interaction between host and pathogenic
microbes that evolved to resist immune responses (13). Functional
impairment (exhaustion) of virus-specific CD8?T cells in chronic
LCMV infection of mice is associated with elevated PD-1 expres-
T cells after HIV infection, and it is associated with T cell
exhaustion and disease progression (15–17). Blockade of the PD-
and restores effector functions, cytokine production, and cell
proliferation. The PD-1–PD-L pathway is also exploited by para-
sites, such as Schistosoma mansoni and Taenia crassiceps, which
suppress effective immune responses by up-regulating PD-Ls on
host macrophages (18, 19). Of further interest, the pathogenic
of PD-L1 results in enhanced T cell proliferation and cytokine
Although the importance of the PD-1–PD-L pathway has been
studied in several infection models, there are no data available
concerning the role of this pathway in fungal infections. In this
study, we report the crucial role of the PD-1–PD-L pathway in a
fungal infection using a mouse model of histoplasmosis. Most
strikingly, PD-1-deficient mice are resistant to lethal challenge with
Hc. During infection, PD-L1 is up-regulated on alveolar and
peritoneal macrophages as well as on all mononuclear cells in the
lungs and on total splenocytes, and PD-L2 is up-regulated on
macrophages and DCs in the lung. The macrophages expressing
upon interaction with CD4?and CD8?T cells in vitro, suggesting
that these macrophages similarly suppress T cell activation in
Author contributions: E.l.-M. and A.G. contributed equally to this work: S.C.A., S.G.N., and
J.D.N. contributed equally to this work; E.L.-M., A.G., S.C.A., S.G.N., and J.D.N. designed
research; E.L.-M. and A.G. performed research; G.J.F. contributed new reagents/analytic
tools; E.L.-M., A.G., G.J.F., S.C.A., S.G.N., and J.D.N. analyzed data; and E.L.-M. wrote the
The authors declare no conflict of interest.
**To whom correspondence may be addressed. E-mail: firstname.lastname@example.org or
This article contains supporting information online at www.pnas.org/cgi/content/full/
© 2008 by The National Academy of Sciences of the USA
February 19, 2008 ?
vol. 105 ?
infected hosts. Furthermore, blockade of the PD-1 pathway in
wild-type mice by administration of monoclonal antibody to PD-1
increased survival by 70%. Our data demonstrate the importance
of the PD-1–PD-L pathway in experimental histoplasmosis and
suggest that this pathway is a target for immunotherapy in his-
toplasmosis and, perhaps, in other fungal diseases.
PD-1-Deficient Mice Survive Lethal Hc Challenge. To study the im-
portance of the PD-1/PD-L pathway in histoplasmosis, groups of
107Hc yeast cells and disease was monitored. In this model of
histoplasmosis, all wild-type mice died by day 25 after infection. In
contrast, 100% of PD-1?/?mice survived, and they were disease
free for ?90 days after infection (Fig. 1a). Moreover, PD-1-
deficient mice also survived challenge with a dose 10 times higher
than the standard lethal dose (data not shown). Initial colony-
106Hc yeast cells were similar between wild-type and PD-1-
deficient mice, showing that the same inoculum was delivered to
both PD-1?/?and wild-type mice. However, in contrast to a steady
increase in the wild-type mice, the pathogen burden rapidly de-
creased in the lungs of PD-1?/?mice, and it could not be detected
developed disseminated disease, as shown by the cfu values ob-
tained from the liver and spleen 3 days after infection (Fig. 1 c and
d). However, by day 13 after infection, PD-1-deficient mice totally
eradicated the Hc.
Histological analysis shows that wild-type mice develop progres-
sive pneumonia, whereas the alveolar spaces of PD-1?/?mice are
mice have bronchointerstitial pneumonia, manifested by edema
as some vascular thrombosis (Fig. 2a). By day 10, the disease in
wild-type mice progresses to necrotizing inflammation with thick-
14, persisting necrotizing pneumonia and fibrosis, as well as loss of
wild-type mice. GMS staining specific for fungal cells shows an
abundance of Hc yeast cells present in the lungs of wild-type mice
(Fig. 2a Insets). In contrast, lungs of the PD-1?/?mice show rare
areas of perivascular inflammation at day 8, GMS staining reveals
no visible organisms (Fig. 2c). At days 10 and 14, multifocal sites of
inflammation were present peribronchially, and lymphocytic infil-
trates were found around some blood vessels, and cfu assays
revealed that the lungs were sterile by day 10. At day 22 after
infection, the lungs of the PD-1-deficient mice look similar to those
Although minor adventitial vasculitis can be detected around some
of the pulmonary vessels, similar findings were present in the lungs
of the mock-infected PD-1-deficient mice. These data clearly show
mice, they can efficiently clear the pathogen and survive high-
Hc Causes Up-Regulation of PD-L1 and PD-L2 Expression on Macro-
phages and Other Cell Types. Because our findings showed that the
lack of a functional PD-1/PD-L pathway abrogated the capacity of
Hc to damage the host, we investigated whether the fungus can
manipulate this inhibitory pathway by influencing the expression
levels of the PD-1 receptor or its ligands, PD-L1 and PD-L2. FACS
analysis showed that PD-L1 was markedly up-regulated on lung
macrophages and dendritic cells, but also on CD4?and CD8?T
cells and, to a lesser extent, on B cells [Fig. 3 a–e and supporting
information (SI) Table 1]. Interestingly, PD-L2 expression was also
up-regulated on a relatively small subset of macrophages (7.3% of
lung macrophages; Fig. 4a) and dendritic cells (13.9% of lung DCs;
Fig. 4b) in the lungs of Hc-infected animals. In contrast, PD-1
expression did not change upon Hc infection on any cell types
latory ligands such as B7–1, B7–2, and ICOS-ligand were not
significantly different in Hc-infected versus control mice (data not
Flow-cytometric analysis of the spleen showed a broad up-
regulation of PD-L1 on total splenocytes in the infected mice (Fig.
Hc yeast cells monitored during a 70-day period,*, P ? 0.0002 (log-rank test).
infected with a sublethal inoculum (5 ? 106) of Hc yeast cells. Each symbol
represents one mouse, and horizontal bars represent median values for each
group. P ? 0.0049 (Kruskal–Wallis test). #, no detectable cfu. Data are repre-
sentative of two independent experiments.
PD-1-deficient mice survive lethal Hc challenge. (a) Survival curves of
La ´za ´r-Molna ´r et al.
February 19, 2008 ?
vol. 105 ?
no. 7 ?
3f). In the spleen, PD-L1 was markedly up-regulated on DCs,
macrophages, and CD4?and CD8?T cells, B cells, and natural
killer (NK) cells as well (SI Fig. 7). When PD-L1 expression levels
of the infection. CD8?and CD4?T cells in the lungs of infected
mice had 30–40% higher PD-L1 expression, compared with the
CD8?and CD4?T cells in the spleen (P ? 0.049 and 0.007, SI
IFN-? is known to be the most important regulator of PD-L1
expression (21). To test its involvement in Hc-induced PD-L
up-regulation, IFN-?-deficient mice were infected and examined
for PD-L expression. CD4?and CD8?T cells in the lungs of
infected IFN-?-deficient mice showed 20–40% lower PD-L1 ex-
pression (P ? 0.009 and 0.049) compared with wild-type mice, and
there was no increase of PD-L1 expression on B cells (P ? 0.009,
SI Table 2). In contrast, there was no increase of PD-L1 expression
on splenocytes of the IFN-?-deficient mice infected with Hc,
suggesting that the up-regulation of PD-L1 on splenocytes depends
PD-L2 was ?2-fold higher in infected IFN-? deficient mice com-
pared with wild-type mice: 13% of macrophages (Fig. 4c) and
21.8% of DCs (Fig. 4d) expressed PD-L2. These data show that
infection with Hc induces an increased expression of PD-Ls that is
also modulated by the availability of cytokines, such as IFN-?.
Macrophages from Hc-infected Mice Suppress T Cell Activation in
Vitro. To investigate whether increased PD-L1 expression on cells
from Hc-infected mice can induce immunosuppression, in vitro T
cell assay was performed. Naı ¨ve CD4?or CD8?T cells purified
from spleens of C57BL/6 mice were activated with low-dose anti-
CD3, and macrophages from control or Hc infected mice were
added to them in different ratios, keeping the T cell number
constant. Peritoneal macrophages that have been shown to up-
regulate PD-L1 expression after Hc infection (SI Fig. 8) were used
for this assay because they can be obtained easily and in reasonable
numbers. Fig. 5 a and b shows that the presence of naı ¨ve macro-
phages greatly increased proliferation of CD4?and CD8?T cells
toplasmosis than wild-type mice. Histological analysis
of the lungs of Hc-infected C57BL/6 mice at days 8 and
14 after infection (a) and mock-infected PD-1?/?(b)
and Hc-infected PD-1?/?mice 8, 14, and 22 days after
infection (c). H&E-stained sections of the lungs are
shown with the Insets representing GMS staining spe-
cific for Hc organisms (shown in black). (Original mag-
PD-1-deficient mice develop less severe his-
nuclear cells and splenocytes of Hc-infected
mice. (a–e) FACS analysis of lung macrophages
(a), dendritic cells (b), CD4?(c) and CD8?T cells
(d), and B cells (e) shows higher levels of PD-L1
expression after Hc infection [uninfected con-
trol mice (Left) and infected mice (Right)]. Val-
ues shown represent geometric mean fluores-
cence intensities for PD-L1, measured for the
gated double-positive cells. Gates were set up
by using the unstained and the appropriate
isotype controls. Representative data of five in-
dependent experiments are shown. (f) PD-L1
up-regulation on splenocytes of Hc-infected
mice depends on the presence of IFN-?. PD-L1
expression on splenocytes of control mice (thin
compared with isotype control antibody
two independent experiments.
PD-L1 is up-regulated on lung mono-
www.pnas.org?cgi?doi?10.1073?pnas.0711918105La ´za ´r-Molna ´r et al.
number of macrophages from Hc-infected mice failed to increase
proliferation of CD4?or CD8?T cells. CD4?T cell proliferation
was inhibited by as much as 53% compared with the control when
infected macrophages were added at a ratio of 1:16 to T cells, the
strongest inhibition being ?80% at the ratio of 1:4. Moreover,
CD8?T cell proliferation was inhibited by ?60% at a 1:32
macrophage-to-T cell ratio, and the highest inhibition was 80% at
a 1:4 ratio. Next we looked at the early and late cytokine responses
elicited by anti-CD3-activated T cells in the presence of macro-
phages from naı ¨ve or Hc-infected mice. Macrophages from naı ¨ve
mice increased anti-CD3-activated secretion of cytokines such as
IL-2 and IFN-? from CD4?T cells (Fig. 5 c and d). However,
addition of macrophages from infected mice substantially de-
creased cytokine production. IL-2 levels were decreased by 75%
and IFN-? levels by 60–80% at 1:16 and 1:4 macrophage-to-T cell
ratios. Macrophages from Hc-infected mice markedly inhibited
cytokine secretion of CD8?T cells as well (SI Fig. 9). These data
suggest that Hc induces a suppressive phenotype of macrophages
activated CD4?and CD8?T cells that could potentially eliminate
Blockade of the PD-1 Pathway Increases Survival of Lethally Infected
renders mice resistant to experimental histoplasmosis, we hypoth-
esized that blockade of this pathway in wild-type mice could be
infected groups of mice with 1.25 ? 107Hc yeast cells and treated
them with either PD-1-blocking antibody or isotype control. The
treatment was started 1 day after infection and consisted of a total
of three doses, one dose given every 3 days. All of the untreated
mice and those receiving the isotype control antibody were dead by
day 12 after infection. However, 70% of the mice treated with the
monoclonal antibody to PD-1 (clone 29F.1A12) survived (Fig. 6).
Importantly, in a repeated experiment continued administration of
the antibodies up to a total of five doses increased survival to 90%
(data not shown). Histological analysis of the lungs of mice from
each group performed 7 days after infection confirmed that,
and dendritic cells in Hc-infected mice. (a and b) PD-L2
on macrophages (a) and dendritic cells (b) of C57BL/6
dendritic cells (d) express PD-L2 in the lungs of IFN-?-
deficient mice infected with Hc. (Left) Data from con-
trol mice. (Right) Data from infected mice. Values
shown are percentages of PD-L2?macrophages from
the total macrophage population. Quadrants were set
both antigens. Data are representative of two inde-
PD-L2 is up-regulated on lung macrophages
cell activation. (a and b) In vitro proliferation of CD4?
(a) and CD8?(b) T cells cocultured with macrophages
from control (open histograms) or Hc infected mice
(hatched histograms). Mean values and SEM are
shown. (c and d) Cytokine production of in vitro-
activated CD4?T cells: IL-2 (c) and IFN-? (d) are signif-
icantly reduced in the presence of macrophages from
Hc-infected mice compared with macrophages from
control mice.*, P ? 0.05;**, P ? 0.01;***, P ? 0.001
(ANOVA, Bonferroni posttest).
Macrophages from Hc-infected mice inhibit T
La ´za ´r-Molna ´r et al.
February 19, 2008 ?
vol. 105 ?
no. 7 ?
generalized bronchointerstitial pneumonia with edema and necro-
sis, anti-PD-1-treated mice had more contained, focal pneumonia
that ultimately resolved (SI Fig. 10). Anti-PD-1-treated mice sur-
vived and were disease free for the follow-up period (?6 months).
Pathogens have developed diverse mechanisms to resist host im-
mune responses. Recent findings suggest that the PD-1/PD-L
pathway plays an important role in the complex interactions be-
tween host and pathogenic microbes. The PD-1/PD-L pathway
mice and humans. PD-1 expression is up-regulated on exhausted
virus-specific T cells causing reversible immune dysfunction and
disease progression in both chronic lymphocytic choriomeningitis
virus (LCMV) infection in mice (14) and HIV infection in humans
(15–17). Blockade of the PD-1/PD-L pathway efficiently restored
the virus-specific effector functions of the exhausted T cells (14–
17). In addition, pathogens such as certain bacteria, protozoa, and
some worms can exploit this pathway to evade host immune
responses (18, 19, 22). Although there is some evidence that the
PD-1/PD-L pathway influences immune responses after acute
infections, this question has not been addressed for fungal disease.
Using a murine model of histoplasmosis, our studies show that
mice that are deficient in PD-1 show 100% survival after high-
inoculum Hc challenge, demonstrating that, in the absence of a
functional PD-1/PD-L activity, the fungus is unable to overcome
host effector responses (Fig. 1a). Importantly, pulmonary and
disseminated disease occur in the PD-1-deficient mice, but the
subsequent immunological responses in these mice and wild-type
animals differ dramatically. The lungs of both wild-type and
however, in the PD-1?/?mice, cfu values rapidly decrease, whereas
they steadily increase in the wild-type mice. Histological studies of
the lungs show that infected PD-1-deficient mice develop patho-
logical findings that are similar but less severe compared with
wild-type mice and that the inflammatory responses in the PD-1-
deficient mice resolve, and the mice survive.
This study shows that the absence of a T cell costimulatory
molecule such as PD-1 confers protection against Hc infection in
mice. In similar studies that used mice deficient in CD40L, another
costimulatory molecule involved in regulating T cell responses and
production of Th1 cytokines there were no differences in survival
or fungal burden (4).
Our findings show that a functional PD-1 pathway is essential for
this fungal pathogen to progressively invade and kill the host. It is
also extremely relevant that the pathogen itself can modulate this
pathway. Our data show a substantial up-regulation of PD-L1 on
in the lungs of Hc-infected mice (SI Table 2), suggesting that other
mechanisms are involved, such as a direct effect of the pathogen or
stimulation from other cytokines released by the infected macro-
phages. PD-L2 up-regulation was also detected on a subset of
macrophages and DCs in the lungs of Hc-infected wild-type mice
(Fig. 4). When IFN-?-deficient mice were infected, there was a
twofold increase in the number of PD-L2-expressing macrophages
and DCs, suggesting that IFN-? has a negative effect on PD-L2
expression (24). However, IFN-? is a key effector cytokine in host
enhances the severity of Hc infection (25, 26). Because of the
complexity of the role of IFN-? in Hc infection, it remains to be
clarified whether the effects observed in the IFN-?-deficient mice
are due to the direct effects of IFN-? or secondary changes in the
we were not able to detect up-regulation of PD-1 on any cell types
percentage of antigen-specific cells or to the transient kinetics of
PD-1 expression in acute Hc infection.
Our data suggest that Hc is able to induce suppression of T cell
responses facilitating its survival within the host. Peritoneal mac-
Fig. 8) significantly inhibited in vitro proliferation and cytokine
production of CD4?and CD8?T cells, whereas control macro-
phages from uninfected mice dose-dependently enhanced anti-
CD3-induced T cell activation (Fig. 5). The inhibition observed
on macrophages from the infected mice. However, we cannot
exclude the role of other mediators, such as nitric oxide, that has
been shown to be involved in inducing a suppressive phenotype in
macrophages (27). Similar studies using other parasites such as
Taenia crassiceps and Schistosoma mansoni have also shown induc-
tion of anergy of naı ¨ve T cells through the selective up-regulation
of PD-L1 and PD-L2 on macrophages (18, 19). Okazaki et al. (12)
suggested a model in which expression of PD-L versus other
costimulatory ligands on DCs would decide the fate of T cell
activation, resulting in either inactivation/anergy or efficient acti-
vation. In accordance with this, in our model, selective increase of
PD-L1 or PD-L2, but not B7–1, B7–2, or ICOSL, on Hc-infected
macrophages induces a suppressive phenotype, in which PD-L
expression will predominate over other ‘‘positive’’ costimulatory
ligands. Macrophages expressing this suppressive phenotype trig-
T cells aimed to eliminate the pathogen, favoring its survival.
PD-L1 up-regulation on other cell types such as CD4?and
CD8?T cells could lead, through an as yet to be identified
mechanism (perhaps involving T cell–T cell signaling; possibly
through the recently identified PD-L1/B7–1 interaction), to a
decrease in the number of CD4?or CD8?T cells that will again
facilitate the survival of the pathogen (28). Based on this model,
blockade of the PD-1 pathway in wild-type mice would enhance
clearance of the pathogen and promote survival of the host,
similar to what occurs in the PD-1?/?mice.
Indeed, our data show that administration of monoclonal anti-
body to PD-1 can efficiently prevent Hc-induced lethality in wild-
strongly suggest that therapeutic targeting of the PD-1 pathway
could be beneficial in the management of histoplasmosis. The
significance of our findings is highlighted by the fact that, despite
intensive therapy with amphotericin B, mortality rates in dissemi-
nated histoplasmosis range from 5% to 10% in immunologically
curves of C57BL/6 mice (n ? 10 for each group) lethally infected with Hc
or saline.*, P ? 0.005 (log-rank test).
Blockade of PD-1 protects mice from lethal histoplasmosis. Survival
www.pnas.org?cgi?doi?10.1073?pnas.0711918105 La ´za ´r-Molna ´r et al.
intact individuals (29, 30) and 46–70% in patients with HIV
with severe disease in individuals infected with HIV (in fact it is an
AIDS-defining infection). Because PD-1 was shown to be up-
HIV, it is reasonable to suggest that blockade of the PD-1 pathway
would benefit the host immune response against both pathogens
through restoration of the antiviral function of CD8?T cells and
abolishing T cell suppression mediated through Hc-infected anti-
gen-presenting cells (APCs). This supposition is supported by the
finding that sublethal Hc infection associated with persistent infec-
tion of LCMV clone 13 resulted in reduced immunity leading to
increased fungal burdens and high mortality (33). Because LCMV
has been shown to cause exhaustion of CD8?T cells through
up-regulation of PD-1 (14), it is intriguing to consider that Hc can
Overall, our studies extend the role of the PD-1/PD-L pathway
in regulating antimicrobial immunity to fungal pathogens by show-
ing that the PD-1/PD-L costimulatory pathway dramatically affects
immune responses against Hc. Blockade of this pathway by anti-
bodies or other pharmacological agents could offer therapeutic
tools in the treatment of histoplasmosis and perhaps other fungal
Materials and Methods
Mice. C57BL/6 mice (6–12 weeks old) were purchased from NCI. PD-1-deficient
mice in the C57BL/6 background were kindly provided by Tasuku Honjo (Kyoto
University) and were used in the experiments at 6–12 weeks of age. IFN-?-
Fungus and Infections. Hc var. capsulatum ATCC 26032 (G217B) was obtained
from the American Type Culture Collection. Infections, cfu counts, and histolog-
ical analysis were performed as described in SI Experimental Procedures.
peritoneal macrophages, from infected and control mice 7 days after infection.
Single-cell suspensions were prepared by collagenase digestion from lungs (34)
and spleens as described in SI Experimental Procedures.
Flow Cytometry. Anti-CD11b-APC, anti-CD19-PE-, FITC-, and APC-conjugated
tibodies for PD-1, PD-L1, and PD-L2 were purchased from eBiosciences, anti-
CD4-PE and anti-CD8-FITC from BD Biosciences. FACS staining and analysis were
performed as described in SI Experimental Procedures.
PD-1 monoclonal antibody (clone 29F.1A12) (35) in PBS was administered i.p.
Ten mice were used for each group.
Statistical Analysis. Statistical analysis was performed as described in SI Experi-
Shared Resource of the Albert Einstein Cancer Center, especially Rani Sellers, for
of the manuscript. We thank T. Honjo (Kyoto University, Kyoto, Japan) and L.
Chen (The Johns Hopkins University) for providing the PD-1/1 mice. E.L.-M. was
supported by a postdoctoral fellowship from the Cancer Research Institute. A.G.
and J.D.N. were supported in part by National Institutes of Health (NIH) Grant
AI056070-01A2. J.D.N. was also partially supported by a Wyeth Vaccine Young
the Center for AIDS Research at the Albert Einstein College of Medicine and
Montefiore Medical Center (NIH Grant AI-51519). S.C.A. and S.G.N. were sup-
ported by NIH Grant AI07289.
1. Deepe GS, Jr (2000) Immune response to early and late Histoplasma capsulatum
infections. Curr Opin Microbiol 3:359–362.
2. Newman SL (1999) Macrophages in host defense against Histoplasma capsulatum.
Trends Microbiol 7:67–71.
memory immunity in pulmonary histoplasmosis. J Immunol 162:7389–7396.
4. Zhou P, Seder RA (1998) CD40 ligand is not essential for induction of type 1 cytokine
responses or protective immunity after primary or secondary infection with His-
toplasma capsulatum. J Exp Med 187:1315–1324.
capsulatum in murine lung. Infect Immun 66:1473–1481.
6. Allen HL, Deepe GS, Jr (2006) B cells and CD4?CD8?T cells are key regulators of the
severity of reactivation histoplasmosis. J Immunol 177:1763–1771.
7. Greenwald RJ, Freeman GJ, Sharpe AH (2005) The B7 family revisited. Annu Rev
8. Dong H, Zhu G, Tamada K, Chen L (1999) B7–H1, a third member of the B7 family,
co-stimulates T-cell proliferation and interleukin-10 secretion. Nat Med 5:1365–1369.
9. Freeman GJ, et al. (2000) Engagement of the PD-1 immunoinhibitory receptor by a
novel B7 family member leads to negative regulation of lymphocyte activation. J Exp
Nat Immunol 2:261–268.
11. Chen L (2004) Co-inhibitory molecules of the B7-CD28 family in the control of T-cell
immunity. Nat Rev Immunol 4:336–347.
12. OkazakiT,HonjoT(2006)ThePD-1–PD-Lpathwayinimmunologicaltolerance. Trends
13. Sharpe AH, Wherry EJ, Ahmed R, Freeman GJ (2007) The function of programmed cell
infection. Nature 439:682–687.
15. Day CL, et al. (2006) PD-1 expression on HIV-specific T cells is associated with T-cell
exhaustion and disease progression. Nature 443:350–354.
16. Petrovas C, et al. (2006) PD-1 is a regulator of virus-specific CD8?T cell survival in HIV
infection. J Exp Med 203:2281–2292.
leads to reversible immune dysfunction. Nat Med 12:1198–1202.
18. Smith P, et al. (2004) Schistosoma mansoni worms induce anergy of T cells via selective
up-regulation of programmed death ligand 1 on macrophages. J Immunol 173:1240–
19. Terrazas LI, Montero D, Terrazas CA, Reyes JL, Rodriguez-Sosa M (2005) Role of the
programmed death-1 pathway in the suppressive activity of alternatively activated
macrophages in experimental cysticercosis. Int J Parasitol 35:1349–1358.
20. Das S, et al. (2006) Expression of B7–H1 on gastric epithelial cells: Its potential role in
regulating T cells during Helicobacter pylori infection. J Immunol 176:3000–3009.
21. Mazanet MM, Hughes CC (2002) B7–H1 is expressed by human endothelial cells and
suppresses T cell cytokine synthesis. J Immunol 169:3581–3588.
22. Liang SC, et al. (2006) PD-L1 and PD-L2 have distinct roles in regulating host immunity
to cutaneous leishmaniasis. Eur J Immunol 36:58–64.
23. Nishimura H, Nose M, Hiai H, Minato N, Honjo T (1999) Development of lupus-like
autoimmune diseases by disruption of the PD-1 gene encoding an ITIM motif-carrying
immunoreceptor. Immunity 11:141–151.
24. Loke P, Allison JP (2003) PD-L1 and PD-L2 are differentially regulated by Th1 and Th2
cells. Proc Natl Acad Sci USA 100:5336–5341.
immunity to infection with Histoplasma capsulatum: TNF-alpha plays a critical role in
maintaining secondary immunity in the absence of IFN-gamma. J Immunol 160:1359–
26. Allendoerfer R, Deepe GS, Jr (1997) Intrapulmonary response to Histoplasma capsu-
latum in gamma interferon knockout mice. Infect Immun 65:2564–2569.
27. MacMicking J, Xie QW, Nathan C (1997) Nitric oxide and macrophage function. Annu
Rev Immunol 15:323–350.
28. Butte MJ, Keir ME, Phamduy TB, Sharpe AH, Freeman GJ (2007) Programmed death-1
ligand 1 interacts specifically with the B7–1 costimulatory molecule to inhibit T cell
responses. Immunity 27:111–122.
29. Assi MA, Sandid MS, Baddour LM, Roberts GD, Walker RC (2007) Systemic histoplas-
mosis: A 15-year retrospective institutional review of 111 patients. Medicine (Balti-
30. Tobon AM, et al. (2005) Disseminated histoplasmosis: A comparative study between
patients with acquired immunodeficiency syndrome and non-human immunodefi-
ciency virus-infected individuals. Am J Trop Med Hyg 73:576–582.
31. Wheat J (1996) Histoplasmosis in the acquired immunodeficiency syndrome. Curr Top
Med Mycol 7:7–18.
32. Wheat LJ, et al. (2000) Factors associated with severe manifestations of histoplasmosis
in AIDS. Clin Infect Dis 30:877–881.
33. Wu-Hsieh BA, et al. (2001) Distinct CD8 T cell functions mediate susceptibility to
histoplasmosis during chronic viral infection. J Immunol 167:4566–4573.
34. Rivera J, Zaragoza O, Casadevall A (2005) Antibody-mediated protection against
Cryptococcus neoformans pulmonary infection is dependent on B cells. Infect Immun
La ´za ´r-Molna ´r et al.
February 19, 2008 ?
vol. 105 ?
no. 7 ?