REVIEWS

Inhibitory B7-family molecules in the

tumour microenvironment
Weiping Zou* and Lieping Chen

Abstract | The B7 family consists of activating and inhibitory co-stimulatory molecules that
positively and negatively regulate immune responses. Recent studies have shown that human
and rodent cancer cells, and stromal cells and immune cells in the cancer microenvironment
upregulate expression of inhibitory B7 molecules and that these contribute to tumour immune
evasion. In this Review, we focus on the roles of these B7 molecules in the dynamic interactions
between tumours and the host immune system, including their expression, regulation and
function in the tumour microenvironment. We also discuss novel therapeutic strategies that
target these inhibitory B7 molecules and their signalling pathways to treat human cancer.
Tumour-associated antigens
(TAAs). Antigens that are
expressed by tumour cells.
These belong to three main
categories: tissuedifferentiation antigens, which
are also expressed by nonmalignant cells; mutated or
aberrantly expressed
molecules; and cancer testis
antigens, which are normally
expressed only by
spermatocytes and
occasionally in the placenta.

Myeloid-derived suppressor
cells
A population of cells that
comprises mature and
immature myeloid cells. They
are expanded and/or activated
during an inflammatory
immune response. Through
direct interactions and
secreted components, they
inhibit Tcell function.

*Department of Surgery,
University of Michigan, Ann
Arbor, Michigan 48109, USA.

Departments of Dermatology
and Oncology, Johns Hopkins
University School of Medicine,
Baltimore, Maryland 21231,
USA.
e-mails: wzou@med.umich.
edu; lchen42@jhmi.edu
doi:10.1038/nri2326

The specific function of an individuals immune system

in different physiological and pathological settings is
regulated by the actions of opposing factors or systems. Common examples of this are the polarization of
Thelper (TH) cells into TH1-cell and TH2-cell subsets
with opposing functions, and the balance between effector Tcell activation and regulatory Tcell activation. At
the molecular level, co-stimulatory members of the B7
family can have both inhibitory and stimulatory effects
on Tcell activation.
An imbalance in immune regulation profoundly
affects tumour-specific Tcell immunity in the cancer
microenvironment and can reshape tumour progression,
metastasis and immunotherapy in patients with cancer1. It
is well known that the lack of naturally induced immunity
specific for tumour-associated antigens (TAAs) is not simply
a passive process whereby adaptive immunity is shielded
from detecting TAAs19. It has been clearly shown that the
tumour microenvironment is comprised of dysfunctional
immune cells that are reprogrammed by active tumourmediated processes to evade tumour-specific immunity in
a highly effective manner. Three important mediators of
this evasion of tumour immunity that have been identified in the tumour microenvironment are: dysfunctional
antigen-presenting cells (APCs), including dendritic cells
(DCs), macrophages1,10 and myeloid-derived suppressor
cells11,12; regulatory T (TReg) cells1316; and high levels of expression of inhibitory B7 molecules by APCs, stromal cells and
tumour cells1,17. The first two mediators have been specifically reviewed in the literature elsewhere1,1013,15. In this
Review, we focus on inhibitory B7 molecules, and detail
their expression, regulation, function and therapeutic
relevance in the tumour microenvironment.

nature reviews | immunology

Inhibitory B7 molecules
T-cell activation and tolerance are not chance occurrences. Many molecules form an orchestrated system to
regulate the interactions between APCs and Tcells. This
interaction is explained by the two-signal model, whereby
signal one is provided to the T-cell receptor of T cells by
the presentation of specific antigens on MHC molecules
expressed by APCs, and signal two is provided by the B7
family and other co-stimulatory molecules that APCs
use to direct and/or fine-tune Tcell responses (FIG.1).
The growing B7 family now comprises seven members,
which are CD80 (also known as B7.1), CD86 (also known
as B7.2), B7-DC (also known as PDL2 or CD273),
B7-H1 (also known as PDL1 or CD274), B7-H2 (also
known as ICOSL), B7-H3 (also known as CD276) and
B7-H4 (also known as B7S1 or B7x)1719. Compelling
evidence indicates that B7 molecules not only provide
crucial positive signals to stimulate and support Tcell
activation, but can also offer negative signals that control
and suppress Tcell responses17,18. These negative signals
are largely provided by the newly identified B7-family
members B7H1 and B7H4.
CD80/CD86CTLA4. CD80 and CD86 control T-cell
activation by binding to and signalling through two
receptors, CD28 and cytotoxic T-lymphocyte antigen 4
(CTLA4), that are expressed by Tcells (FIG.1). CD80
and CD86 are not classically considered as inhibitory
B7 molecules. However, on Tcell activation, the expression of their inhibitory receptor, CTLA4, is induced
on Tcells, and engagement of CTLA4 by CD80 and
CD86 can limit and decrease Tcell activation. The
role of CTLA4 in controlling Tcell activation and its
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REVIEWS
APC

T cell

CD80

CD28

CD86

CTLA4

MHC

TCR

B7-DC

?
PD-1

B7-H1

CD80

B7-H2

ICOS

B7-H3

B7-H4

?
IgV-like domain

Regulatory T (TReg) cells

A Tcell population that can

functionally suppress an
immune response by
influencing the activity
of another cell type.
Several phenotypically
distinct regulatory Tcell
populations exist. The classic
regulatory Tcells are
CD4+CD25+FOXP3+ Tcells
known as TReg cells.

IgC-like domain

Figure 1 | The B7 family and antigen presentation to

Tcells. Antigen-presenting cells (APCs) or APC-like cells
Nature
Reviews |to
Immunology
present a specific antigen on MHC
molecules
the Tcell
receptor (TCR) of Tcells. Members of the B7 family and
other co-stimulatory molecules are used to direct and/or
fine-tune Tcell responses. The newly identified B7H1
and B7H4 molecules provide negative signals that
control and suppress Tcell responses. Human tumour
cells and tumour-associated APCs express limited levels
of the stimulatory B7-family members CD80 and CD86,
and high levels of the inhibitory B7-family members
B7H1 and B7H4. This imbalance between the expression
of stimulatory and inhibitory B7 molecules might
contribute to tumour immune evasion in the tumour
microenvironment. CTLA4; cytotoxic T-lymphocyte
antigen 4; ICOS, inducible T-cell co-stimulator; PD-1,
programmed cell death1.

therapeutic relevance have been reviewed elsewhere19

and are not discussed in detail here. CTLA4-specific
antibodies have recently entered clinical trials for the
treatment of various human cancers19.

Two-signal model
The concept that both the
MHCpeptide complex
(signal1) and co-stimulatory
signals delivered by B7-family
molecules expressed by
antigen-presenting cells
(signal2) are required for Tcell
activation. The absence of
signal 2 results in the induction
of Tcell anergy or deletion.

Cytotoxic Tlymphocyteantigen 4
(CTLA4). After engagement by
CD80 or CD86 on antigenpresenting cells, CTLA4
signalling in activated Tcells
induces cell-cycle arrest,
decreases cytokine production
and inhibits Tcell responses.
CD4+CD25+ TReg cells
constitutively express CTLA4.

B7H1. B7H1 was first cloned on the basis of its DNA

sequence homology to other B7 molecules belonging
to the immunoglobulin superfamily20. Programmed
cell death 1 (PD1; also known as CD279) has been
subsequently identified as a counter-receptor for B7H1
(Ref.21) (FIG.1), and B7H1 is therefore also known as
PDL1 to emphasize this receptorligand interaction21.
However, experimental evidence indicates that additional counter-receptor(s) other than PD1 can mediate
the functions of B7H1. In the absence of PD1 or if
binding to PD1 is blocked, B7H1 can have a stimulatory effect on Tcell immunity17,20,2224. Moreover, CD80
is an additional counter-receptor for B7H1 for the
inhibition of Tcell responses25. To make the situation
more complicated, B7H1 can also function as a receptor
to transmit signals into Tcells26 and tumour cells27. In
summary, B7H1 can act as both ligand and receptor to
execute immunoregulatory functions.

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B7H4. B7H4 was also identified by DNA sequence

homology to other B7 molecules2830. B7H4 remains an
orphan ligand, although evidence indicates that a receptor can be induced and could function on Tcells28,30,31.
The currently known functions of B7H4 are exclusively
inhibitory and the effect of B7H4 might be mediated
by a single receptor. B- and T-lymphocyte attenuator
(BTLA) was initially proposed to be the receptor for
B7H4 (Ref.32), but recent studies show that this is not
the case and that herpes virus entry mediator is the
ligand for BTLA3335.

Expression pattern
CD80 and CD86 have a restricted expression pattern,
being expressed mainly by professional APCs and
haematopoietic cells, but rarely by stromal cells and
nonhaematopoietic cancer cells17,18. By contrast, mRNA
encoding B7H1 and B7H4 is found in almost all tissues
and most stromal and haematopoietic cells. This distribution pattern indicates unique functions for these molecules in both lymphoid and non-lymphoid organs.
Expression of B7H1. The expression of mRNA encoding B7H1 is abundant in many tissues and organs
in humans20,36 and mice21. A recent study indicates
that the phosphatase and tensin homologue (PTEN)
phosphatidylinositol-3kinase pathway might be
important in the post-transcriptional regulation of
B7H1 cell-surface expression in tumours37. B7H1
protein is often expressed by activated cells including
Tcells, Bcells, DCs, monocytes/macrophages 20,21,23,
natural killer (NK) cells38, activated vascular endothelial cells39, mesenchymal stem cells40 and cultured bonemarrow-derived mast cells41. B7H1 is also found to
be expressed constitutively in immune-privileged sites
including the eyes and placenta42,43, which indicates
that B7H1 might inhibit self-reactive Tcells or Bcells
and therefore control inflammatory responses in these
tissues and organs.
Most human cancers tested so far express high levels
of B7H1 protein44 (TABLE1). However, low or rare B7H1
expression is observed in most mouse and human
tumour-cell lines44. This might be owing to the lack
of the complete cancer microenvironment in cell lines
invitro as these tumour-cell lines can upregulate B7H1
protein expression in response to cytokines44. Another
possibility, albeit less likely, is that certain molecular
profiles have been altered in established tumour-cell
lines during culture. Therefore, the results obtained
from established tumour-cell lines invitro might
need to be interpreted carefully. In addition to tumour
cells, B7H1 protein expression has been observed
in human tumour-associated DCs 23,45, fibroblasts 46
and Tcells47,48.
Expression of B7H4. Similar to B7H1, mRNA encoding
B7H4 is widely distributed in peripheral tissues28,49,50.
However, although the expression of B7H4 cell-surface
protein was detected in normal human epithelial cells of
the female genital tract, kidney, lung and pancreas, B7H4
protein was generally absent in other normal human
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REVIEWS
Table 1 | B7H1 expression in human cancers and its immunological, clinical and pathological associations*
Human cancer type

B7H1+ cases/ Immunological, clinical and pathological associations

total cases

Refs

Breast cancer

24/56

Number of B7H1+ Tcells correlates with tumour size, stage and HER2 expression

Colon cancer

16/25

ND

36,44

Gastric cancer

45/105

B7H1 expression correlates with increased tumour size, metastasis and poor survival

36,89

Glioma

10/10

B7H1 expression by tumour cells inhibits Tcell activation invitro

Leukaemia

17/30

B7H1 expression by leukaemia cells has no effect on Tcell activation invitro

Lung cancer

86/87

B7H1+ regions of the tumour contain fewer Tcells in non-small cell lung cancer

Melanoma

22/22

ND

Multiple myeloma

82/82

B7H1+ plasma cells inhibit Tcell activation invitro

Oesophageal cancer

18/41

B7H1 expression is associated with poor prognosis

Ovarian cancer

82/93

B7H1 expression by tumour cells is associated with a decreased number of Tcells in

the tumour and poor prognosis. B7H1+ tumour-associated dendritic cells inhibit Tcell
function.

Pancreatic cancer

20/51

B7H1 expression by tumour cells is associated with a decreased number of tumour

Tcells and poor prognosis.

36,44,48

125
81
36,44,86
44
126
88

Peripheral Tcell lymphoma 7/11

ND

Renal-cell carcinoma

130/196

B7H1 expression by tumour cells is associated with poor prognosis

Thymic neoplasm

28/34

ND

Urothelial cancer

142/268

B7H1 expression by tumour cells is associated with advanced stage, recurrence and
poor survival

23,36,44,87

127
36
44,85,128
36
129,130

*The data for each tumour type are assembled from more than one report. Some data have not been included in the table if less than 10 tumour samples were
reported for a particular type of human cancer. HER2 (also known as ERBB2 or Neu) is a tumour-associated antigen that is expressed in about 25% of cases of breast
cancer. Vaccines against HER2 are being tested for cancer therapy in humans. Monoclonal antibodies specific for HER2 (such as trastuzumab (Herceptin;
Genentech/Roche)) are approved for the therapy of human breast cancer. ND, not determined.

Myeloid DCs
A subset of dendritic cells (DCs)
that are lineage-negative,
HLA-DR+CD11c+ (in humans)
mononuclear cells with a
monocytoid appearance.
Human myeloid DCs might be
derived from myeloid
precursors (for example,
monocytes, macrophages and
CD11c+ precursors).

Plasmacytoid DCs
A subset of dendritic cells (DCs)
that are lineage negative,
HLA-DR+CD11c (in humans)
mononuclear cells with a
microscopic appearance
similar to plasmablasts.
Plasmacytoid DCs are the main
producers of typeI interferon.

somatic tissues50. Nonetheless one study did observe

broad cell-surface expression of B7H4 protein on mouse
haematopoietic cells30. The cause of this interspecies
difference is unknown.
B7H4 is commonly detectable in the human cancer
microenvironment (TABLE2). For example, human ovarian cancers express high levels of B7H4 protein31,49,5154,
and low levels of soluble B7H4 protein were found in
the sera from patients with ovarian cancer52,53. In addition to tumour cells, tumour-infiltrating macrophages31,55
and endothelial cells of small blood vessels56 in the cancer microenvironment are also found to constitutively
express B7H4.
In summary, the current data reveal broad expression
patterns of B7H1 and B7H4 protein in human tumours,
in contrast to their rare expression in normal tissues. These
data indicate that post-transcriptional regulation could
have a crucial role in the control of B7H1 and B7H4
protein expression in normal tissues and organs, and
that this regulatory mechanism is aberrant in tumours.
Further investigation of the regulatory mechanisms and
the signalling pathways leading to expression of B7H1
and B7H4 will generate important information for the
understanding of tumour immune evasion and provide
potential molecular targets for treating human cancers.

Regulation of expression
The tumour microenvironment contains a large number of
cytokines and inflammatory mediators1,57,58. Some of these
molecules can regulate the expression of B7H1 and B7H4.

nature reviews | immunology

Regulation of B7H1 expression. B7H1 expression can

be induced or maintained by many cytokines23,36,44, of
which interferon (IFN) is the most potent. Established
human tumour-cell lines rarely express B7H1 protein
on the cell surface, but high levels of B7H1 expression
can be induced by treatment with IFN in most of the
cell lines tested so far44. Consistent with this, IFN can
induce high levels of B7H1 expression by normal epithelial cells, vascular endothelial cells39, proximal tubular
epithelial cells59 and myeloid DCs36,44. It is assumed that a
strong TH1-cell response can induce B7H1 expression
by APCs and other cells through IFN, and in turn maintain the threshold of Tcell activation to avoid tissue and
organ damage. In addition to IFN, typeI IFN can also
stimulate B7H1 expression by hepatocytes60, monocytes,
DCs61 and tumour cells (S. Wei, I. Kryczek, L. Chen and
W. Zou, unpublished observations). In this context, after
virus infection, tumour-associated plasmacytoid DCs
produce large amounts of typeI IFN invitro62, which
can in turn induce B7H1 expression23. Plasmacytoid
DCs preferentially induce TH2-cell responses in certain
situations63. Therefore, the expression of B7H1 could
potentially be stimulated in both TH1- and TH2-cellbiased conditions, although the expression and relevance
of B7H1 expression in TH1- or TH2-cell-associated diseases remains to be tested.
In light of its stimulatory effect on B7H1 expression, IFN could thus be a double-edged sword in
tumour immunity. Whereas IFN could increase antigen processing and presentation by upregulating the
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Table 2 | B7H4 expression in human cancers and its immunological, clinical and pathological associations*
Human cancer type

B7H4+ cases/ Immunological, clinical and pathological associations

total cases

Refs

Breast cancer (primary)

165/173

B7H4 expression is associated with lack of expression of progesterone receptor and HER2

50,54

Breast cancer
(metastatic)

240/246

ND

Lung cancer

35/86

B7H4 expression is more common in patients with lymph-node metastasis

Ovarian cancer

202/216

B7H4 tumour-associated macrophages inhibit Tcell activation and predict poor survival

31,49,5155

Prostate cancer

120/823

B7H4 expression by tumour cells is associated with disease spread, recurrence and death

90

Renal-cell carcinoma

153/259

B7H4 expression by tumour cells is associated with poor survival

Uterine endometrioid
adenocarcinoma

90/90

B7H4 expression by tumour cells is associated with weak Tcell infiltration and high-risk
tumours

50

49,131

56
132

*The data for each tumour type are assembled from more than one report. Some data have not been included in the table if less than 10 tumour samples were
reported for a particular type of human cancer. HER2 (also known as ERBB2 or Neu) is a tumour-associated antigen that is expressed in about 25% of cases of breast
cancer. Vaccines against HER2 are being tested for cancer therapy in humans. Monoclonal antibodies specific for HER2 (such as trastuzumab (Herceptin;
Genentech/Roche)) are approved for the therapy of human breast cancer. ND, not determined.

Indoleamine 2,3dioxygenase
(IDO). An intracellular haemcontaining enzyme that
catalyses the oxidative
catabolism of tryptophan.
Insufficient availability of
tryptophan can lead to Tcell
apoptosis and anergy.

Arginase
An enzyme that converts
larginine into lornithine and
urea.

Danger signals
Agents that alert the immune
system to stress, usually by
interacting with Toll-like
receptors and other patternrecognition receptors, and
thereby promote the
generation of innate and
adaptive immune responses.
Danger signals can be
associated with microbial
invaders (exogenous danger
signals) or produced by
damaged cells (endogenous
danger signals).

expression of MHC molecules and components of the

antigen-processing machinery64, the effects of IFN on
B7H1 expression might downregulate Tcell immunity. This could explain, at least partially, why IFN
has not been effective as a therapeutic agent for most
human cancers. In addition, IFN has been shown to
stimulate the expression of indoleamine 2,3-dioxygenase
(IDO)65 and arginase6668 on APCs. Such APCs could
suppress anti-tumour immune responses. Therefore,
it is not surprising that an increase in the number of
TAA-specific cytotoxic T lymphocytes (CTLs) does not
always translate into clinical regression of cancers6972.
In addition, IFN was also found to mediate CD4+
Tcell loss and impair secondary anti-tumour immune
responses after successful initial immunotherapy in
tumour-bearing mouse models73. As B7H1 expression
can be induced on APCs and multiple human epithelial tumours, stimulation of B7H1 expression could be
a strategy used by the tumour to evade Tcellmediated
tumour immunity.
Regulation of B7H4 expression. The regulation of
B7H4 expression has only been studied in the human
system. Interleukin6 (IL6) and IL10 stimulate B7H4
expression by monocytes, macrophages and myeloid
DCs. The DCdifferentiation cytokines, GMCSF
(granulocyte/macrophage colony-stimulating factor)
and IL4, decrease B7H4 expression by these cells
induced by IL6 and IL10 (Refs31,55,74) (FIG.2). IFNs
seem to have a minimal effect on the induction of B7H4
expression, in contrast to the induction of B7H1 expression44. In human ovarian cancer, tumour-associated TReg
cells trigger macrophages to produce IL6 and IL10, and
these cytokines in turn stimulate B7H4 expression by
APCs in an autocrine and/or paracrine manner55. High
levels of IL6 and IL10, but not GMCSF and IL4, are
detected in the ovarian tumour microenvironment.
Therefore, this dysfunctional cytokine network in the
tumour microenvironment enables APCs to express
B7H4. Interestingly, IL4, IL6, IL10 and GMCSF
have no regulatory effects on B7H4 expression on
tumour cells, which indicates that B7H4 on tumour

470 | june 2008 | volume 8

cells and B7H4 on APCs might be functionally distinct

and be differentially regulated31,55. Collectively, B7H1
and B7H4 are regulated by distinct mechanisms.
These differential regulatory patterns have important
implications in the generation and amplification of
tumour-specific immunity.

Evasion of tumour immunity

The physiological functions of inhibitory B7-family
members are to limit, terminate and attenuate Tcell
responses, by which they prevent Tcell hyperactivation
and avoid tissue and organ damage during immune
responses17,18. B7H1-deficient75,76 and B7H4deficient77
mice have been generated and reported in the published literature. The inhibitory B7 molecules can be
induced in response to inflammation and potentially
to broader danger signals. It is therefore possible that
the expression of these molecules might contribute
to denovo cancer initiation and development. At the
present time, however, there is no evidence that these
molecules participate in carcinogenesis. However, these
inhibitory B7 molecules could suppress ongoing or
induced tumour immunity.
B7H1 expression by tumour cells. Initial studies
showed that transgenic expression of B7H1 on a B7
H1deficient mouse P815 mastocytoma cell line did not
change its tumourigenicity in naive mice44. However,
B7H1+ P815 tumour cells could continue to grow in
the presence of adoptively transferred P815-specific
Tcells, which are sufficient to induce the regression of
wild-type P815 tumours44. This finding is consistent
with the observation that transfection of B7H1 into
a highly immunogenic P815 variant, which regresses
spontaneously in naive mice, led to progressive growth
of this line44. Similarly, in the presence of potent Tcell
immunity, B7H1+ tumours are much more resistant
to Tcell-mediated destruction in several mouse models7880 as well as in a human Tcell leukaemia model81.
Interestingly, several B7H1 mouse tumours start to
express B7H1 during growth invivo. Injection of an
antibody specific for B7H1 increased Tcell immunity
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REVIEWS

APC

TReg cell
IL-6
and IL-10
GM-CSF and IL-4
B7-H4

Cell cycle
T cell

Figure 2 | B7H4+ antigen-presenting cells in the

tumour microenvironment. Nature
B7H4+Reviews
antigen-presenting
| Immunology
cells (APCs), such as tumour-associated macrophages,
induce Tcell cycle arrest. B7H4 expression can be induced
by interleukin-6 (IL6) and IL10, and is inhibited by the
dendritic-cell differentiation cytokines granulocyte/
macrophage colony-stimulating factor (GM-CSF) and IL4.
Regulatory T (TReg) cells can trigger IL6 and IL10
production by APCs, and in turn upregulate B7H4
expression by APCs. APCs that have been conditioned by
TReg cells inhibit Tcell function through B7H4. High levels
of TReg cells, and IL6 and IL10 are found in the tumour
microenvironment, which helps to explain the observation
of B7H4+ APCs in this environment.

and induced tumour regression82. In these experimental

settings, it could not be excluded that B7H1 expression
by host cells, especially immune cells, could also have a
role. In this regard, PD1-deficient mice79,83 were shown
to have increased Tcell immunity and were more resistant to the outgrowth of transplanted murine tumours,
which indicates that host-derived B7H1 has a negative
effect on tumour immunity. In addition, recent studies
show that the expression of B7H1 could also function as a receptor to transmit an anti-apoptotic signal
to cancer cells as a means to resist immune-mediated
destruction27 (see later).
B7H1 expression by host cells. Myeloid DCs in human
tumours and tumour-draining lymph nodes express
high levels of B7H1 (Refs23,45) . The interaction
between B7H1+ myeloid DCs and tumourassociated
Tcells leads to the downregulation of expression of
myeloid-DC-derived IL12 and upregulation of expression of the immunosuppressive cytokine IL10 by
myeloid DCs in a B7H1-dependent manner. Blockade
of B7H1 on tumour-infiltrating myeloid DCs increases
IFN production by Tcells and promotes tumour infiltration by IFN-producing Tcells. Adoptive transfer of
such Tcells improves the clearance of human tumours
in xenotransplanted mice 23. These data support the
idea that B7H1 has a role in the downregulation of
DCmediated tumour immunity.
In the DA1-3b/C3H mouse model of acute myeloid leukaemia, dormant tumour cells resist CTLmediated killing
owing to high levels of B7H1 expression by tumour cells84.
nature reviews | immunology

In this model, the injection of irradiated acute myeloid

leukaemia cells transduced by CXCchemokine ligand
10 (CXCL10) led to prophylactic immunity in mice.
Interestingly, CXCL10 stimulated B7H1 expression by
NK cells, and NKcell-associated B7H1 was essential
for this prophylactic immunity38. These data indicate
that a stimulatory counterreceptor for B7H1 is
expressed by Tcells in the dormant tumour state owing
to the unique tumour microenvironment. It remains to
be determined if this mechanism is operative in other
tumour models and in patients with cancer.
B7H4. Although high levels of B7H4 expression
are observed in human cancers (TABLE2), there are no
published data showing that the expression of B7H4
by cancer cells can lead to accelerated tumour growth
or progression in immune-competent animals. The
immunopathological relevance of B7H4 has only
been studied in patients with ovarian cancer. High
levels of B7H4 expression are found on a population of tumour-associated macrophages in patients
with ovarian carcinoma. B7H4+ macrophages inhibit
TAA-specific Tcell effector function31. These findings
show that B7H4 is a negative regulator of TAA-specific
Tcell immunity and is a molecular target for tumour
immunotherapy.
In summary, B7H1 and B7H4 are selectively
expressed by various cellular components in the tumour
microenvironment, where they can inhibit tumourspecific Tcell immunity.

Cancer progression
Clinical data have documented that the expression of
inhibitory B7 molecules correlates with poor prognosis of various types of human cancer (TABLES1,2).
However, all of these studies are retrospective and cancer tissues were evaluated for the expression of these
molecules at a specific time point. As it is unknown
whether the level of expression of inhibitory B7 molecules varies during disease progression, the conclusions and the implications from these analyses should
be carefully considered. Nevertheless, these studies
represent an important step towards understanding
the prognosis of and developing novel treatments for
patients with cancer.
High levels of expression of B7H1 were initially
reported in many human tumours44. Subsequent studies confirmed and extended these observations (TABLE1).
Frozen47 and formalin-fixed85 tumour tissues of clear-cell
renal carcinoma stained with a human B7H1-specific
monoclonal antibody44 showed that B7H1 expression
is an indicator of poor prognosis for patient survival47.
In this study, however, the expression of B7H1 by
tumour-infiltrating lymphocytes derived from a fraction of patients was also significantly associated with
poor prognosis47. Some reports have shown that B7H1
expression on tumour cells might also be associated with
decreased numbers of tumour-infiltrating lymphocytes
in patients with cancer86,87. Similarly, B7H1 expression
was also identified as a poor prognostic factor in patients
with oesophageal, gastric88,89 and ovarian87 cancers.
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The broad expression pattern of B7H1 in cancer
microenvironments and its relationship with clinical and
pathological parameters in patients with cancer provide
strong evidence that B7H1 is pathologically relevant,
and that B7H1 and its signalling pathway are justified
targets for treating human cancer.
Although the role of B7H4 expression by tumour
cells in evading tumour immunity has not been well
established, several human studies indicate that B7H4
expression might be associated with the progression of
certain types of human cancer. In patients with renal
carcinoma56 and prostate cancer90, B7H4 expression by
tumour cells is associated with adverse clinical features.
A recent study in patients with ovarian cancer showed
that the level of B7H4 expression by tumour-associated
macrophages, but not tumour cells, correlates with the
number of TReg cells in the tumour. Further, expression
of B7H4 by macrophages and the number of TReg cells
are both negatively associated with patient outcome55. In
this context, B7H4 is shown to be a signature gene that
is consistently expressed in breast cancer91.

Mechanisms of action
B7H1-expressing cells use at least six distinct mechanisms to evade Tcell immunity (FIG.3): inducing apoptosis, anergy or exhaustion of Tcells, forming a molecular
shield to protect tumour cells from lysis, inducing production of the immunosuppressive cytokine IL10 and
promoting TReg-cell-mediated suppression. It is possible
that these mechanisms work as a hierarchy to evade
immunity at different levels of Tcell function to impose
tight control.

Anergy
A state of non-responsiveness
to antigen. Anergic T or Bcells
cannot respond to their
cognate antigens under
optimal conditions of
stimulation.

Exhaustion
An operational definition that
refers to the loss of antigenspecific Tcell responses invivo
after prolonged or repetitive
stimulation with antigen. This
has been best observed in a
model of infection with
lymphocytic choriomeningitis
virus Docile strain, for which
the exact mechanism is still not
understood.

Apoptosis. Co-culture with B7H1+ tumour-cell lines

increased the apoptosis of human TAA-specific Tcells;
blocking B7H1 decreased this apoptosis44. Subsequent
studies showed that B7H1-mediated apoptosis is a
common physiological process used by the host to
maintain homeostasis in peripheral tissues. For example,
B7H1-deficient mice have an accumulation of CD8+
Tcells in the liver due to decreased apoptosis75. Liver
Kupffer cells92 and stellate cells93 constitutively express
B7H1, and human hepatocytes express low levels
of B7H1 (Ref.60). This ready availability of B7H1 in
the liver might explain the decreased apoptosis of Tcells
in B7H1-deficient mice75. B7H1 is also constitutively
expressed in the eye. After corneal allografting, B7H1+
corneal endothelium interacted with PD1+CD4+ Tcells
and led to Tcell apoptosis43. This observation could at
least partially explain how the immune-privileged status
of the cornea could be established.
B7H1 could have a role in the induction of effector Tcell apoptosis in both a PD1-dependent and
-independent manner. In one human Tcell clone, B7H1
can induce apoptosis of PD1 Tcells44, but in most mouse
models, PD1 is required for B7H1mediated apoptosis17.
However, B7H1 can bind to PD1 (Ref.21) and CD80
(Ref.25), and thereby regulate Tcell function. The detailed
molecular mechanisms of apoptosis mediated by B7H1
remain to be elucidated, and could occur indirectly or
through an uncharacterized direct pathway.

472 | june 2008 | volume 8

Anergy. The role of B7H1 in Tcell anergy was determined with the use of a cell-culture system in which
alloreactive Tcells were induced by monocyte-derived
DCs. IL10-treated DCs induced unresponsive Tcells
similar to anergic Tcells, but these Tcells could be
rescued by restimulation with DCs treated with an
antibody specific for human B7H1 (Ref.94). A subsequent study showed that resting DCinduced tolerance
of CD8 + Tcells in mixed bone-marrow chimaeras
could be prevented by the ablation of PD1+ Tcells95.
In the non-obese diabetic (NOD) mouse model, B7H1
and PD1, but not CTLA4 and TReg cells, were shown
to be crucial for Tcell tolerance and anergy96. Given
the inducible nature of B7H1 expression in peripheral
tissues and of PD1 expression by activated Tcells, it
was assumed that effector Tcells, after priming in the
lymph nodes, migrate to peripheral tissues, where they
are induced to express PD1, and become functionally
anergic when engaged with B7H1 on peripheral organs
through PD1. However, this view was challenged by
two recent studies97,98. These studies have shown that the
expression of PD1 can be induced on Tcells while they
are still in lymphoid organs during priming. Blockade of
B7H1 or PD1 by neutralizing monoclonal antibodies
before T-cell egress to peripheral organs could convert
anergic Tcells into fully activated effector Tcells97,98.
Therefore, the interaction of B7H1 with PD1 could
regulate Tcell anergy in both priming and effector
phases of an immune response. In the context of tumour
immune pathology, human tumour-associated DCs
highly express B7H1 (Refs23,45) and PD1+ Tcells99
are found in the tumours. It remains unknown whether
the B7H1PD1 interaction induces anergy of human
TAA-specific naive and effector Tcells.
Exhaustion. This mechanism was revealed in studies of chronic infection. PD1 is highly expressed by
functionally exhausted antigen-specific Tcells, and
B7H1 could be detected in infected tissues and lymphoid organs. Blockade of B7H1 or PD1 can rescue
the functionality of these exhausted Tcells100103. The
data indicate that PD1 might transmit an inhibitory
signal that dominates TCR signalling and decreases
Tcell responses during prolonged exposure to antigens
during chronic infection.
Cancer could be considered as a chronic inflammatory disease58. Not only do up to 15% of cancers
worldwide have a direct infectious origin104, but many
human tumours are also related to chronic irritation and
inflammation1. Human tumour-associated DCs highly
express B7H1, and blocking B7H1 markedly increases
the effector function of tumour Tcells23. In this context,
a recent study showed that PD1 is upregulated on a significant fraction of tumour-infiltrating Tcells in patients
with melanoma and that blockade of PD1 increased
TAA-specific Tcell proliferation and function99. Taken
together, these data indicate that B7H1 and PD1 could
result in human tumour-specific Tcell exhaustion. This
notion, although it has not been formally tested in cancer, needs to be taken into consideration when designing
tumour immunotherapies.
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REVIEWS

IL-10 production
PD-1
Anergy

Exhaustion

Apoptosis

Immune suppression
B7-H1

TReg cell

APC

B7-H1
Tumour cell

Molecular
shield
Protected from
cytotoxic lysis

CTL

Figure 3 | The inhibitory actions of B7H1 in tumour immune evasion. Tumour cells
and tumour-associated antigen-presenting cells (APCs) express high levels of B7H1. The
NatureinReviews
Immunology
potential suppressive mechanisms of B7H1 have been addressed
various| models
invitro and invivo, and it is probable that a combination of mechanisms, rather than a
single mode of action, is used by B7H1-expressing cells. B7H1+ tumour cells and APCs
might induce Tcell apoptosis, anergy, functional exhaustion or IL10 production. B7H1+
tumour cells might be resistant to lysis by cytotoxic T lymphocytes (CTLs); this has been
described as a molecular shield. Tumour-associated regulatory T (TReg) cells might express
B7H1 and mediate Tcell suppression partially through the B7H1PD1 pathway. The
detailed molecular mechanisms remain poorly understood.

Molecular shield. The inclusion of neutralizing monoclonal antibodies specific for B7H1 or PD1 often
increases TAA-specific CTL-mediated lysis or cytokine
release against murine and human tumour cells that
express B7H1 (Refs8284,105,106). This observation indicates that endogenous levels of expression of
B7H1 might be sufficient to confer resistance against
CTLmediated lysis. The effect of neutralizing monoclonal
antibodies specific for B7H1 or PD1 varies depending on the system, ranging from moderate to dramatic.
For example, after transfection to express high levels of
B7H1, P815 mouse tumour cells are much more resistant to lysis by antigen-specific CD8+ Tcells82. Similarly,
the addition of a B7H1-specific monoclonal antibody to
B7H1transfected B16F10 melanoma cells results in the
increased secretion of cytokines from 2C CTLs105. This
resistance to cytotoxic lysis mediated by the expression of
high levels of B7H1 depends on the B7H1PD1 interaction and was described as being a molecular shield82.
In addition, tumour-specific Tcells are more effective at
lysing human glioma cells that express wild-type PTEN
with low-level B7H1 expression than those that express
mutant PTEN with high-level B7H1 expression37. The
nature reviews | immunology

acquisition of lysis resistance by tumour cells occurs

rapidly, within 416 hours, and was also observed for
allogeneic27,105 and re-directed Tcells37 in addition to TAAspecific Tcells. This effect could be interpreted in terms
of the rapid induction of suppressive effects on Tcells
through interaction with PD1. However, further studies showed that this was due to the unique nature of the
tumour-cell surface, but not to the dysfunction of Tcells,
because wild-type tumour cells in the presence of B7H1+
tumour cells in the same culture could still be lysed by
Tcells82. By disabling the intracellular domain of B7H1
on cancer cells, the molecular shield is eliminated and
the tumourcells become susceptible invitro to immune
mediated destruction. Importantly, tumours expressing
B7H1 with a truncated intracellular domain also become
more susceptible to Tcell-mediated immunotherapy
invivo compared with tumours expressing wild-type
B7H1. By contrast, the intracellular truncation of PD1
on Tcells does not eliminate the molecular-shield effect27.
In addition, B7H1 reverse signalling in cancer cells also
renders resistance to apoptosis mediated by antibodies
specific for CD95 (also known as FAS) and by staurosporin
toxin from Streptomyces staurospores (a broad-spectrum
protein-kinase inhibitor)27. In light of its broad expression pattern, B7H1 might be a ubiquitous anti-apoptotic
receptor expressed by cancer cells and might have a
general role in the resistance of tumour cells to immune
destruction and other apoptosis-based therapies.
IL10 production. Selective induction of IL10 was found
initially following the stimulation of human Tcells with
CD3-specific antibody and B7H1immunoglobulin
fusion protein20. Tumour-associated B7H1+ DCs also
induce Tcell production of IL10 (Ref.23). Subsequent
studies have established a correlation between the upregulation of B7H1 expression and increased levels of IL10
in patients with HIV and HBV infection107,108. It remains
to be determined whether increased IL10 production
has an important role in B7H1-mediated suppression
of Tcell responses invivo.
TReg-cell-mediated suppression. It has been suggested that
B7H1 is involved in the development and function of
TReg cells. B7H1+ vascular endothelial cells109 and gastric
epithelial cells110 induce the development of TReg cells in
an invitro culture system. However, it has not been shown
that B7H1 or PD1 is required for TReg-cell differentiation invivo. In tumour-bearing mice, TReg cells activated
by IDO+ DCs induce B7H1 expression on target DCs.
Importantly, the ability of these TReg cells to suppress
Tcell activation could be abrogated by antibody specific
for B7H1 (Ref.111). In support of this observation, it has
been shown that human tumour-associated DCs express
B7H1 and mediate Tcell suppression in a B7H1dependent manner23,45. By contrast, in non-Hodgkin lymphoma, intratumoral CD4+CD25+ TReg cells can express
B7H1, and blocking B7H1 with a monoclonal antibody
partially decreases TReg-cell-mediated Tcell inhibition112.
These results indicate that B7H1 expression by tumourassociated DCs and TReg cells might contribute to tumour
immune suppression.
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REVIEWS
B7H4 can also evade tumour immunity. B7H4 has
been studied in less detail than B7H1 in the context of
tumour immune evasion, but there is evidence indicating that B7H4 might exert its function through myeloid
APCs and TReg cells to mediate Tcell suppression in the
tumour microenvironment31,55,74. Tumourassociated
macrophages markedly outnumber other types of APC,
such as DCs, and form an abundant population of APCs in
solid tumours113116. One population of ovariancancerassociated macrophages expresses high levels of B7H4,
and these B7H4+ macrophages induce Tcell cycle arrest
invitro and invivo partially through B7H4 (Ref.31).
These findings indicate that B7H4 might contribute to
tumour immune evasion, and that B7H4 is a molecular
target for tumour immunotherapy.
In human ovarian cancer, TReg cells and B7H4+
macrophages are co-localized and their numbers are
correlated in the tumour environment16,31,55,74. TReg cells
trigger high levels of IL6 and IL10 production by APCs,
and in turn, these cytokines stimulate the expression
of B7H4 by APCs, which renders the APCs immuno
suppressive55 (FIG.2). These findings are in line with the
observations that macrophages spontaneously produce
IL6 and IL10 in the ovarian tumour environment55,62.
These data mechanistically link IL10, B7H4, TReg cells
and APCs, and provide a new cellular and molecular
mechanism for TReg-cell-mediated immunosuppression
at the level of APCs13,55 (FIG.2).

Implications for cancer immunotherapy

Many tumour-associated APCs and tumour cells express
B7H1 and B7H4, and these molecules can mediate
Tcell suppression. The manipulation of B7-induced
immune suppression might therefore be a broadly applicable therapeutic modality to treat human cancers.

Small interfering RNA

Double-stranded RNAs
(dsRNAs) with sequences that
precisely match a given gene
are able to knock down the
expression of that gene by
directing RNA-degrading
enzymes to destroy the
encoded mRNA transcript. The
two most common forms of
dsRNAs used for gene silencing
are short usually 21-bp long
small interfering RNAs
(siRNAs) or the plasmiddelivered short hairpin RNAs
(shRNAs).

Antisense oligonucleotides
Short, gene-specific sequences
of nucleic acids that are
of the opposite strand
(complementary) to the
targeted mRNA. Classical
antisense oligonucleotides
target specific strands of RNA
within a cell, thereby
preventing translation of these
RNAs.

B7H1 and PD1 blockade. Preclinical data have set the

stage for clinical trials by blocking B7H1 in patients with
cancer. In the context of its suppressive effects on Tcells
and anti-apoptotic effect on tumour cells, it would seem
beneficial to block B7H1 using neutralizing antibodies.
However, antibodies specific for B7H1 could potentially
also block the interaction of B7H1 with a putative costimulatory receptor. In this regard, it has been reported
that local expression of B7H1 can provide positive costimulation for naive Tcells and promote organ-specific
autoimmunity and transplant rejection in mice117. For
obvious reasons, therapeutic antibodies should be tested
for their ability to block the B7H1CD80 interaction in
addition to blocking B7H1PD1 binding. Despite the
possible immune-stimulatory roles of B7H1 in some
mouse models of transplantation and autoimmune
diseases, the effects of B7H1 are largely immunosuppressive on mouse and human tumour immunity. In
terms of the suppressive effects of B7H1, blocking
B7H1 could possibly enhance ongoing autoimmune
diseases. For example, treatment with B7H1-specific
antibody moderately accelerates experimental autoimmune encephalomyelitis in mice118. B7H1-deficient
NOD mice also develop diabetes more rapidly than wildtype NOD mice119, and the loss of B7H1 expression on

474 | june 2008 | volume 8

non-haematopoietic cells is crucial for the accelerated

disease120. However, in light of the mild autoimmune
phenotypes in B7H1-deficient mice75,76, B7H1 blockade will be unlikely to result in the generation of severe
autoimmune disease.
In addition to the effects of B7H1 on transplantation,
autoimmune diseases and tumours, it has been found
that trophoblasts, which are located in the maternal
fetal interface, express B7H1, and B7H1+ trophoblasts
might have a role in the suppression of maternalfetal
immunity42. Therefore, a potential side effect of B7H1
blockade by antibody in pregnant women could be the
termination of pregnancy. Nevertheless, B7H1-deficient
mice produce normal size litters, which indicates that
this is also a less likely event.
Similarly, neutralizing antibodies specific for PD1
will be an important addition to clinical trials. However,
neutralizing antibodies specific for PD1 might block the
interaction of PD1 with B7-DC (FIG.1), another counterreceptor for PD1 with potentially stimulatory effects. In
addition, PD1-specific antibodies might increase the
risk of severe autoimmune diseases, as predicted from
the spontaneous development of autoimmune diseases
in PD1-deficient mice17,18. Finally, although invitro
assays support the claim that certain specific monoclonal
antibodies are antagonistic, if appropriate crosslinking is
provided, these antibodies could be potentially agonistic121. Nonetheless, the significant potential benefits of
the clinical application of B7H1PD1 blockade warrant
extensive experimental and clinical studies in patients
with cancer.
B7H4 blockade. Efficient neutralizing antibodies specific
for human B7H4 are not yet available. Small interfering RNA
(siRNA)54 and antisense oligonucleotides specific for B7H4
(Refs31,74) have been used to block B7H4 expression.
Blocking the expression of B7H4 by tumourassociated
macrophages disables their suppressive capacity, enables
TAA-specific effector Tcell function and reduces tumour
growth in human ovarian cancer xenografts31,74. Taken
together with the proposed inhibitory role for B7H4 in
mouse systems2830, these data justify B7H4 as a promising new target for development of therapeutic reagents for
the treatment of human cancers.
Combinatorial blockade. It is probable that multiple
suppressive mechanisms mediate immune evasion in a
given tumour. Combinatorial treatments will therefore
be required to reverse tumour immune-escape pathways
and lead to potent TAA-specific Tcell immunity. In
tumour-bearing mouse models, for example, blocking
B7H1 in combination with blocking TGF signalling
using neutralizing antibodies synergistically induces
tumour regression133. B7H1 expression is induced
on tumour-associated TReg cells in non-Hodgkin lymphoma and on target DCs in murine tumourdraining
lymph nodes. These TReg cells inhibit the function of
PD1+ tumour-infiltrating Tcells, partially through the
B7H1PD1 pathway111,112. These studies indicate that
simultaneously blocking B7H1PD1 and TReg cells
might be therapeutically relevant.
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REVIEWS
The administration of a blocking monoclonal antibody specific for B7H1 increases the therapeutic effects
of a co-stimulatory CD137-specific agonistic monoclonal
antibody82,122 and of tumour-cell vaccination123. B7H1
and PD1 blockade together with an HSP70 vaccine124
can increase Tcell immunity and decrease tumour
growth and metastasis in mouse models. In addition to
these examples, blocking the B7H1PD1 pathway in
combination with blockade of other well-defined suppressive molecules, including CTLA4, VEGF, B7H4,
TGF, arginase or IDO1, could be possible options in
preclinical and clinical settings to treat cancer.

Concluding remarks
There is now compelling evidence to show that tumours
escape host immunity by actively developing multiple
suppressive mechanisms in the tumour microenvironment. The mechanisms that underlie the interactions
between the immune system and the tumour microenvironment, particularly in humans, are a crucial and
understudied area of cancer immunology, the understanding of which will have a significant impact on
the success of immunotherapy strategies. The selective
expression of inhibitory B7 molecules in the tumour
microenvironment has been determined as an important immunosuppressive mechanism in many types
of human tumour. Therefore, the manipulation of the
expression of and signalling through these molecules is
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Acknowledgements

We would like to thank our former and current trainees and

collaborators for their intellectual input and hard work. The
work described in this Review was supported by grants from
the United States National Institutes of Health and the United
States Department of Defense.

DATABASES
Entrez Gene: http://www.ncbi.nlm.nih.gov/entrez/query.
fcgi?db=gene
B7-DC | B7-H1 | B7-H2 | B7-H3 | B7-H4 | CD80 | CD86

FURTHER INFORMATION
Weiping Zous homepage:
http://sitemaker.umich.edu/zou.lab/home
Lieping Chens homepage:
http://gradimmunology.med.som.jhmi.edu
All links are active in the online pdf

volume 8 | june 2008 | 477

2008 Nature Publishing Group