inhibitory b7 family molecules in tumour microenvironment

The specific function of an individual’s immune system in different physiological and pathological settings is regulated by the actions of opposing factors or sys-tems. Common examples of this are the polarization of T helper (TH) cells into TH1-cell and TH2-cell subsets with opposing functions, and the balance between effec-tor T-cell activation and regulatory T-cell activation. At the molecular level, co-stimulatory members of the B7 family can have both inhibitory and stimulatory effects on T-cell activation.

An imbalance in immune regulation profoundly affects tumour-specific T-cell 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 TAAs1–9. It has been clearly shown that the tumour microenvironment is comprised of dysfunctional immune cells that are reprogrammed by active tumour-mediated processes to evade tumour-specific immunity in a highly effective manner. Three important mediators of this evasion of tumour immunity that have been identi-fied 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) cells13–16; and high levels of expres-sion of inhibitory B7 molecules by APCs, stromal cells and tumour cells1,17. The first two mediators have been spe-cifically reviewed in the literature elsewhere1,10–13,15. In this Review, we focus on inhibitory B7 molecules, and detail their expression, regulation, function and therapeutic relevance in the tumour microenvironment.

Inhibitory B7 moleculesT-cell activation and tolerance are not chance occur-rences. Many molecules form an orchestrated system to regulate the interactions between APCs and T cells. 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 T-cell 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 PD-L2 or CD273), B7-H1 (also known as PD-L1 or CD274), B7-H2 (also known as ICOSL), B7-H3 (also known as CD276) and B7-H4 (also known as B7S1 or B7x)17–19. Compelling evidence indicates that B7 molecules not only provide crucial positive signals to stimulate and support T-cell activation, but can also offer negative signals that control and suppress T-cell responses17,18. These negative signals are largely provided by the newly identified B7-family members B7-H1 and B7-H4.

CD80/CD86–CTLA4. 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 T cells (FIG. 1). CD80 and CD86 are not classically considered as inhibitory B7 molecules. However, on T-cell activation, the expres-sion of their inhibitory receptor, CTLA4, is induced on T cells, and engagement of CTLA4 by CD80 and CD86 can limit and decrease T-cell activation. The role of CTLA4 in controlling T-cell activation and its

*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: [email protected]; [email protected]:10.1038/nri2326

Tumour-associated antigens(TAAs). Antigens that are expressed by tumour cells. These belong to three main categories: tissue-differentiation antigens, which are also expressed by non-malignant 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 cellsA 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 T-cell function.

Inhibitory B7-family molecules in the tumour microenvironmentWeiping 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.

nATuRe RevIewS | immunology vOLuMe 8 | june 2008 | 467

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Nature Reviews | Immunology

CD80

CD80

CD86

MHC

B7-DC

B7-H1

B7-H2

B7-H3

B7-H4

CD28

CTLA4

PD-1

?

?

?

ICOS

TCR

APC T cell

IgV-like domain IgC-like domain

Regulatory T (TReg) cellsA T-cell population that can functionally suppress an immune response by influencing the activity of another cell type. Several phenotypically distinct regulatory T-cell populations exist. The classic regulatory T cells are CD4+CD25+FOXP3+ T cells known as TReg cells.

Two-signal modelThe concept that both the MHC–peptide complex (signal 1) and co-stimulatory signals delivered by B7-family molecules expressed by antigen-presenting cells (signal 2) are required for T-cell activation. The absence of signal 2 results in the induction of T-cell anergy or deletion.

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

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.

B7‑H1. B7-H1 was first cloned on the basis of its DnA sequence homology to other B7 molecules belonging to the immunoglobulin superfamily20. Programmed cell death 1 (PD-1; also known as CD279) has been subsequently identified as a counter-receptor for B7-H1 (ReF. 21) (FIG. 1), and B7-H1 is therefore also known as PD-L1 to emphasize this receptor–ligand interaction21. However, experimental evidence indicates that addi-tional counter-receptor(s) other than PD-1 can mediate the functions of B7-H1. In the absence of PD-1 or if binding to PD-1 is blocked, B7-H1 can have a stimula-tory effect on T-cell immunity17,20,22–24. Moreover, CD80 is an additional counter-receptor for B7-H1 for the inhibition of T-cell responses25. To make the situation more complicated, B7-H1 can also function as a receptor to transmit signals into T cells26 and tumour cells27. In summary, B7-H1 can act as both ligand and receptor to execute immunoregulatory functions.

B7‑H4. B7-H4 was also identified by DnA sequence homology to other B7 molecules28–30. B7-H4 remains an orphan ligand, although evidence indicates that a recep-tor can be induced and could function on T cells28,30,31. The currently known functions of B7-H4 are exclusively inhibitory and the effect of B7-H4 might be mediated by a single receptor. B- and T-lymphocyte attenuator (BTLA) was initially proposed to be the receptor for B7-H4 (ReF. 32), but recent studies show that this is not the case and that herpes virus entry mediator is the ligand for BTLA33–35.

Expression patternCD80 and CD86 have a restricted expression pattern, being expressed mainly by professional APCs and haematopoietic cells, but rarely by stromal cells and non-haematopoietic cancer cells17,18. By contrast, mRnA encoding B7-H1 and B7-H4 is found in almost all tissues and most stromal and haematopoietic cells. This distri-bution pattern indicates unique functions for these mol-ecules in both lymphoid and non-lymphoid organs.

Expression of B7‑H1. The expression of mRnA encod-ing B7-H1 is abundant in many tissues and organs in humans20,36 and mice21. A recent study indicates that the phosphatase and tensin homologue (PTen)– phosphatidylinositol-3-kinase pathway might be important in the post-transcriptional regulation of B7-H1 cell-surface expression in tumours37. B7-H1 protein is often expressed by activated cells including T cells, B cells, DCs, monocytes/macrophages20,21,23, natural killer (nK) cells38, activated vascular endothe-lial cells39, mesenchymal stem cells40 and cultured bone-marrow-derived mast cells41. B7-H1 is also found to be expressed constitutively in immune-privileged sites including the eyes and placenta42,43, which indicates that B7-H1 might inhibit self-reactive T cells or B cells and therefore control inflammatory responses in these tissues and organs.

Most human cancers tested so far express high levels of B7-H1 protein44 (TABLe 1). However, low or rare B7-H1 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 in vitro as these tumour-cell lines can upregulate B7-H1 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 in vitro might need to be interpreted carefully. In addition to tumour cells, B7-H1 protein expression has been observed in human tumour-associated DCs23,45, fibroblasts46 and T cells47,48.

Expression of B7‑H4. Similar to B7-H1, mRnA encoding B7-H4 is widely distributed in peripheral tissues28,49,50. However, although the expression of B7-H4 cell-surface protein was detected in normal human epithelial cells of the female genital tract, kidney, lung and pancreas, B7-H4 protein was generally absent in other normal human

Figure 1 |TheB7familyandantigenpresentationtoTcells.Antigen-presenting cells (APCs) or APC-like cells present a specific antigen on MHC molecules to the T-cell receptor (TCR) of T cells. Members of the B7 family and other co-stimulatory molecules are used to direct and/or fine-tune T-cell responses. The newly identified B7-H1 and B7-H4 molecules provide negative signals that control and suppress T-cell 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 B7-H1 and B7-H4. 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 death 1.

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Myeloid DCsA 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 DCsA 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 type I interferon.

somatic tissues50. nonetheless one study did observe broad cell-surface expression of B7-H4 protein on mouse haematopoietic cells30. The cause of this interspecies difference is unknown.

B7-H4 is commonly detectable in the human cancer microenvironment (TABLe 2). For example, human ovar-ian cancers express high levels of B7-H4 protein31,49,51–54, and low levels of soluble B7-H4 protein were found in the sera from patients with ovarian cancer52,53. In addi-tion to tumour cells, tumour-infiltrating macrophages31,55 and endothelial cells of small blood vessels56 in the can-cer microenvironment are also found to constitutively express B7-H4.

In summary, the current data reveal broad expression patterns of B7-H1 and B7-H4 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 B7-H1 and B7-H4 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 B7-H1 and B7-H4 will generate important information for the understanding of tumour immune evasion and provide potential molecular targets for treating human cancers.

Regulation of expressionThe tumour microenvironment contains a large number of cytokines and inflammatory mediators1,57,58. Some of these molecules can regulate the expression of B7-H1 and B7-H4.

Regulation of B7‑H1 expression. B7-H1 expression can be induced or maintained by many cytokines23,36,44, of which interferon-γ (IFnγ) is the most potent. established human tumour-cell lines rarely express B7-H1 protein on the cell surface, but high levels of B7-H1 expression can be induced by treatment with IFnγ in most of the cell lines tested so far44. Consistent with this, IFnγ can induce high levels of B7-H1 expression by normal epi-thelial cells, vascular endothelial cells39, proximal tubular epithelial cells59 and myeloid DCs36,44. It is assumed that a strong TH1-cell response can induce B7-H1 expression by APCs and other cells through IFnγ, and in turn main-tain the threshold of T-cell activation to avoid tissue and organ damage. In addition to IFnγ, type I IFn can also stimulate B7-H1 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 type I IFn in vitro62, which can in turn induce B7-H1 expression23. Plasmacytoid DCs preferentially induce TH2-cell responses in certain situations63. Therefore, the expression of B7-H1 could potentially be stimulated in both TH1- and TH2-cell-biased conditions, although the expression and relevance of B7-H1 expression in TH1- or TH2-cell-associated dis-eases remains to be tested.

In light of its stimulatory effect on B7-H1 expres-sion, IFnγ could thus be a ‘double-edged sword’ in tumour immunity. whereas IFnγ could increase anti-gen processing and presentation by upregulating the

Table 1 | B7‑H1 expression in human cancers and its immunological, clinical and pathological associations*

Humancancertype B7‑H1+cases/totalcases

immunological,clinicalandpathologicalassociations Refs

Breast cancer 24/56 Number of B7-H1+ T cells correlates with tumour size, stage and HER2‡ expression 36,44,48

Colon cancer 16/25 ND 36,44

Gastric cancer 45/105 B7-H1 expression correlates with increased tumour size, metastasis and poor survival 36,89

Glioma 10/10 B7-H1 expression by tumour cells inhibits T-cell activation in vitro 125

Leukaemia 17/30 B7-H1 expression by leukaemia cells has no effect on T-cell activation in vitro 81

Lung cancer 86/87 B7-H1+ regions of the tumour contain fewer T cells in non-small cell lung cancer 36,44,86

Melanoma 22/22 ND 44

Multiple myeloma 82/82 B7-H1+ plasma cells inhibit T-cell activation in vitro 126

Oesophageal cancer 18/41 B7-H1 expression is associated with poor prognosis 88

Ovarian cancer 82/93 B7-H1 expression by tumour cells is associated with a decreased number of T cells in the tumour and poor prognosis. B7-H1+ tumour-associated dendritic cells inhibit T-cell function.

23,36,44,87

Pancreatic cancer 20/51 B7-H1 expression by tumour cells is associated with a decreased number of tumour T cells and poor prognosis.

127

Peripheral T-cell lymphoma 7/11 ND 36

Renal-cell carcinoma 130/196 B7-H1 expression by tumour cells is associated with poor prognosis 44,85,128

Thymic neoplasm 28/34 ND 36

Urothelial cancer 142/268 B7-H1 expression by tumour cells is associated with advanced stage, recurrence and poor survival

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.

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Indoleamine 2,3-dioxygenase(IDO). An intracellular haem-containing enzyme that catalyses the oxidative catabolism of tryptophan. Insufficient availability of tryptophan can lead to T-cell apoptosis and anergy.

ArginaseAn enzyme that converts l-arginine into l-ornithine and urea.

Danger signalsAgents that alert the immune system to stress, usually by interacting with Toll-like receptors and other pattern-recognition 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 IFnγ on B7-H1 expression might downregulate T-cell immu-nity. This could explain, at least partially, why IFnγ has not been effective as a therapeutic agent for most human cancers. In addition, IFnγ has been shown to stimulate the expression of indoleamine 2,3-dioxygenase (IDO)65 and arginase66–68 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 cancers69–72. In addition, IFnγ was also found to mediate CD4+ T-cell loss and impair secondary anti-tumour immune responses after successful initial immunotherapy in tumour-bearing mouse models73. As B7-H1 expression can be induced on APCs and multiple human epithe-lial tumours, stimulation of B7-H1 expression could be a strategy used by the tumour to evade T-cell-mediated tumour immunity.

Regulation of B7‑H4 expression. The regulation of B7-H4 expression has only been studied in the human system. Interleukin-6 (IL-6) and IL-10 stimulate B7-H4 expression by monocytes, macrophages and myeloid DCs. The DC-differentiation cytokines, GM-CSF (granulocyte/macrophage colony-stimulating factor) and IL-4, decrease B7-H4 expression by these cells induced by IL-6 and IL-10 (ReFS 31,55,74) (FIG. 2). IFns seem to have a minimal effect on the induction of B7-H4 expression, in contrast to the induction of B7-H1 expres-sion44. In human ovarian cancer, tumour-associated TReg cells trigger macrophages to produce IL-6 and IL-10, and these cytokines in turn stimulate B7-H4 expression by APCs in an autocrine and/or paracrine manner55. High levels of IL-6 and IL-10, but not GM-CSF and IL-4, are detected in the ovarian tumour microenvironment. Therefore, this dysfunctional cytokine network in the tumour microenvironment enables APCs to express B7-H4. Interestingly, IL-4, IL-6, IL-10 and GM-CSF have no regulatory effects on B7-H4 expression on tumour cells, which indicates that B7-H4 on tumour

cells and B7-H4 on APCs might be functionally distinct and be differentially regulated31,55. Collectively, B7-H1 and B7-H4 are regulated by distinct mechanisms. These differential regulatory patterns have important implications in the generation and amplification of tumour-specific immunity.

Evasion of tumour immunityThe physiological functions of inhibitory B7-family members are to limit, terminate and attenuate T-cell responses, by which they prevent T-cell hyperactivation and avoid tissue and organ damage during immune responses17,18. B7-H1-deficient75,76 and B7-H4-deficient77 mice have been generated and reported in the pub-lished 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 de novo 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.

B7‑H1 expression by tumour cells. Initial studies showed that transgenic expression of B7-H1 on a B7- H1-deficient mouse P815 mastocytoma cell line did not change its tumourigenicity in naive mice44. However, B7-H1+ P815 tumour cells could continue to grow in the presence of adoptively transferred P815-specific T cells, which are sufficient to induce the regression of wild-type P815 tumours44. This finding is consistent with the observation that transfection of B7-H1 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 T-cell immunity, B7-H1+ tumours are much more resistant to T-cell-mediated destruction in several mouse mod-els78–80 as well as in a human T-cell leukaemia model81. Interestingly, several B7-H1– mouse tumours start to express B7-H1 during growth in vivo. Injection of an antibody specific for B7-H1 increased T-cell immunity

Table 2 | B7‑H4 expression in human cancers and its immunological, clinical and pathological associations*

Humancancertype B7‑H4+cases/totalcases

immunological,clinicalandpathologicalassociations Refs

Breast cancer (primary) 165/173 B7-H4 expression is associated with lack of expression of progesterone receptor and HER2‡ 50,54

Breast cancer (metastatic)

240/246 ND 50

Lung cancer 35/86 B7-H4 expression is more common in patients with lymph-node metastasis 49,131

Ovarian cancer 202/216 B7-H4+ tumour-associated macrophages inhibit T-cell activation and predict poor survival 31,49,51–55

Prostate cancer 120/823 B7-H4 expression by tumour cells is associated with disease spread, recurrence and death 90

Renal-cell carcinoma 153/259 B7-H4 expression by tumour cells is associated with poor survival 56

Uterine endometrioid adenocarcinoma

90/90 B7-H4 expression by tumour cells is associated with weak T-cell infiltration and high-risk tumours

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.

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APC TReg cell

IL-6 and IL-10

GM-CSF and IL-4

↑ B7-H4

Cell cycle

T cell

and induced tumour regression82. In these experimental settings, it could not be excluded that B7-H1 expression by host cells, especially immune cells, could also have a role. In this regard, PD-1-deficient mice79,83 were shown to have increased T-cell immunity and were more resist-ant to the outgrowth of transplanted murine tumours, which indicates that host-derived B7-H1 has a negative effect on tumour immunity. In addition, recent studies show that the expression of B7-H1 could also func-tion as a receptor to transmit an anti-apoptotic signal to cancer cells as a means to resist immune-mediated destruction27 (see later).

B7‑H1 expression by host cells. Myeloid DCs in human tumours and tumour-draining lymph nodes express high levels of B7-H1 (ReFS 23,45). The interaction between B7-H1+ myeloid DCs and tumour-associated T cells leads to the downregulation of expression of myeloid-DC-derived IL-12 and upregulation of expres-sion of the immunosuppressive cytokine IL-10 by myeloid DCs in a B7-H1-dependent manner. Blockade of B7-H1 on tumour-infiltrating myeloid DCs increases IFnγ production by T cells and promotes tumour infil-tration by IFnγ-producing T cells. Adoptive transfer of such T cells improves the clearance of human tumours in xenotransplanted mice23. These data support the idea that B7-H1 has a role in the downregulation of DC-mediated tumour immunity.

In the DA1-3b/C3H mouse model of acute myeloid leu-kaemia, dormant tumour cells resist CTL-mediated killing owing to high levels of B7-H1 expression by tumour cells84.

In this model, the injection of irradiated acute myeloid leukaemia cells transduced by CXC-chemokine ligand 10 (CXCL10) led to prophylactic immunity in mice. Interestingly, CXCL10 stimulated B7-H1 expression by nK cells, and nK-cell-associated B7-H1 was essential for this prophylactic immunity38. These data indicate that a stimulatory counter-receptor for B7-H1 is expressed by T cells 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.

B7‑H4. Although high levels of B7-H4 expression are observed in human cancers (TABLe 2), there are no published data showing that the expression of B7-H4 by cancer cells can lead to accelerated tumour growth or progression in immune-competent animals. The immunopathological relevance of B7-H4 has only been studied in patients with ovarian cancer. High levels of B7-H4 expression are found on a popula-tion of tumour-associated macrophages in patients with ovarian carcinoma. B7-H4+ macrophages inhibit TAA-specific T-cell effector function31. These findings show that B7-H4 is a negative regulator of TAA-specific T-cell immunity and is a molecular target for tumour immunotherapy.

In summary, B7-H1 and B7-H4 are selectively expressed by various cellular components in the tumour microenvironment, where they can inhibit tumour- specific T-cell immunity.

Cancer progressionClinical data have documented that the expression of inhibitory B7 molecules correlates with poor prog-nosis of various types of human cancer (TABLeS 1,2). However, all of these studies are retrospective and can-cer 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 mol-ecules varies during disease progression, the conclu-sions 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 B7-H1 were initially reported in many human tumours44. Subsequent stud-ies confirmed and extended these observations (TABLe 1). Frozen47 and formalin-fixed85 tumour tissues of clear-cell renal carcinoma stained with a human B7-H1-specific monoclonal antibody44 showed that B7-H1 expression is an indicator of poor prognosis for patient survival47. In this study, however, the expression of B7-H1 by tumour-infiltrating lymphocytes derived from a frac-tion of patients was also significantly associated with poor prognosis47. Some reports have shown that B7-H1 expression on tumour cells might also be associated with decreased numbers of tumour-infiltrating lymphocytes in patients with cancer86,87. Similarly, B7-H1 expression was also identified as a poor prognostic factor in patients with oesophageal, gastric88,89 and ovarian87 cancers.

Figure 2 |B7‑H4+antigen‑presentingcellsinthetumourmicroenvironment.B7-H4+ antigen-presenting cells (APCs), such as tumour-associated macrophages, induce T-cell cycle arrest. B7-H4 expression can be induced by interleukin-6 (IL-6) and IL-10, and is inhibited by the dendritic-cell differentiation cytokines granulocyte/macrophage colony-stimulating factor (GM-CSF) and IL-4. Regulatory T (TReg) cells can trigger IL-6 and IL-10 production by APCs, and in turn upregulate B7-H4 expression by APCs. APCs that have been conditioned by TReg cells inhibit T-cell function through B7-H4. High levels of TReg cells, and IL-6 and IL-10 are found in the tumour microenvironment, which helps to explain the observation of B7-H4+ APCs in this environment.

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AnergyA state of non-responsiveness to antigen. Anergic T or B cells cannot respond to their cognate antigens under optimal conditions of stimulation.

ExhaustionAn ‘operational’ definition that refers to the loss of antigen-specific T-cell responses in vivo 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.

The broad expression pattern of B7-H1 in cancer microenvironments and its relationship with clinical and pathological parameters in patients with cancer provide strong evidence that B7-H1 is pathologically relevant, and that B7-H1 and its signalling pathway are justified targets for treating human cancer.

Although the role of B7-H4 expression by tumour cells in evading tumour immunity has not been well established, several human studies indicate that B7-H4 expression might be associated with the progression of certain types of human cancer. In patients with renal carcinoma56 and prostate cancer90, B7-H4 expression by tumour cells is associated with adverse clinical features. A recent study in patients with ovarian cancer showed that the level of B7-H4 expression by tumour-associated macrophages, but not tumour cells, correlates with the number of TReg cells in the tumour. Further, expression of B7-H4 by macrophages and the number of TReg cells are both negatively associated with patient outcome55. In this context, B7-H4 is shown to be a signature gene that is consistently expressed in breast cancer91.

Mechanisms of actionB7-H1-expressing cells use at least six distinct mecha-nisms to evade T-cell immunity (FIG. 3): inducing apop-tosis, anergy or exhaustion of T cells, forming a molecular shield to protect tumour cells from lysis, inducing pro-duction of the immunosuppressive cytokine IL-10 and promoting TReg-cell-mediated suppression. It is possible that these mechanisms work as a hierarchy to evade immunity at different levels of T-cell function to impose tight control.

Apoptosis. Co-culture with B7-H1+ tumour-cell lines increased the apoptosis of human TAA-specific T cells; blocking B7-H1 decreased this apoptosis44. Subsequent studies showed that B7-H1-mediated apoptosis is a common physiological process used by the host to maintain homeostasis in peripheral tissues. For example, B7-H1-deficient mice have an accumulation of CD8+ T cells in the liver due to decreased apoptosis75. Liver Kupffer cells92 and stellate cells93 constitutively express B7-H1, and human hepatocytes express low levels of B7-H1 (ReF. 60). This ready availability of B7-H1 in the liver might explain the decreased apoptosis of T cells in B7-H1-deficient mice75. B7-H1 is also constitutively expressed in the eye. After corneal allografting, B7-H1+ corneal endothelium interacted with PD-1+CD4+ T cells and led to T-cell apoptosis43. This observation could at least partially explain how the immune-privileged status of the cornea could be established.

B7-H1 could have a role in the induction of effec-tor T-cell apoptosis in both a PD-1-dependent and -independent manner. In one human T-cell clone, B7-H1 can induce apoptosis of PD-1– T cells44, but in most mouse models, PD-1 is required for B7-H1-mediated apoptosis17. However, B7-H1 can bind to PD-1 (ReF. 21) and CD80 (ReF. 25), and thereby regulate T-cell function. The detailed molecular mechanisms of apoptosis mediated by B7-H1 remain to be elucidated, and could occur indirectly or through an uncharacterized direct pathway.

Anergy. The role of B7-H1 in T-cell anergy was deter-mined with the use of a cell-culture system in which alloreactive T cells were induced by monocyte-derived DCs. IL-10-treated DCs induced unresponsive T cells similar to anergic T cells, but these T cells could be rescued by restimulation with DCs treated with an antibody specific for human B7-H1 (ReF. 94). A subse-quent study showed that resting DC-induced tolerance of CD8+ T cells in mixed bone-marrow chimaeras could be prevented by the ablation of PD-1+ T cells95. In the non-obese diabetic (nOD) mouse model, B7-H1 and PD-1, but not CTLA4 and TReg cells, were shown to be crucial for T-cell tolerance and anergy96. Given the inducible nature of B7-H1 expression in peripheral tissues and of PD-1 expression by activated T cells, it was assumed that effector T cells, after priming in the lymph nodes, migrate to peripheral tissues, where they are induced to express PD-1, and become functionally anergic when engaged with B7-H1 on peripheral organs through PD-1. However, this view was challenged by two recent studies97,98. These studies have shown that the expression of PD-1 can be induced on T cells while they are still in lymphoid organs during priming. Blockade of B7-H1 or PD-1 by neutralizing monoclonal antibodies before T-cell egress to peripheral organs could convert anergic T cells into fully activated effector T cells97,98. Therefore, the interaction of B7-H1 with PD-1 could regulate T-cell anergy in both priming and effector phases of an immune response. In the context of tumour immune pathology, human tumour-associated DCs highly express B7-H1 (ReFS 23,45) and PD-1+ T cells99 are found in the tumours. It remains unknown whether the B7-H1–PD-1 interaction induces anergy of human TAA-specific naive and effector T cells.

Exhaustion. This mechanism was revealed in stud-ies of chronic infection. PD-1 is highly expressed by functionally exhausted antigen-specific T cells, and B7-H1 could be detected in infected tissues and lym-phoid organs. Blockade of B7-H1 or PD-1 can rescue the functionality of these exhausted T cells100–103. The data indicate that PD-1 might transmit an inhibitory signal that dominates TCR signalling and decreases T-cell responses during prolonged exposure to antigens during chronic infection.

Cancer could be considered as a chronic inflam-matory 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 B7-H1, and blocking B7-H1 markedly increases the effector function of tumour T cells23. In this context, a recent study showed that PD-1 is upregulated on a sig-nificant fraction of tumour-infiltrating T cells in patients with melanoma and that blockade of PD-1 increased TAA-specific T-cell proliferation and function99. Taken together, these data indicate that B7-H1 and PD-1 could result in human tumour-specific T-cell exhaustion. This notion, although it has not been formally tested in can-cer, needs to be taken into consideration when designing tumour immunotherapies.

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Anergy

IL-10 production

TReg cell

ApoptosisImmune suppression

Molecular shield

CTL

B7-H1

Exhaustion

APC

Protected fromcytotoxic lysis

PD-1

Tumour cell

B7-H1

Molecular shield. The inclusion of neutralizing mono-clonal antibodies specific for B7-H1 or PD-1 often increases TAA-specific CTL-mediated lysis or cytokine release against murine and human tumour cells that express B7-H1 (ReFS 82–84,105,106). This observa-tion indicates that endogenous levels of expression of B7-H1 might be sufficient to confer resistance against CTL-mediated lysis. The effect of neutralizing monoclonal antibodies specific for B7-H1 or PD-1 varies depend-ing on the system, ranging from moderate to dramatic. For example, after transfection to express high levels of B7-H1, P815 mouse tumour cells are much more resist-ant to lysis by antigen-specific CD8+ T cells82. Similarly, the addition of a B7-H1-specific monoclonal antibody to B7-H1-transfected B16-F10 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 B7-H1 depends on the B7-H1–PD-1 inter-action and was described as being a ‘molecular shield’82. In addition, tumour-specific T cells are more effective at lysing human glioma cells that express wild-type PTen with low-level B7-H1 expression than those that express mutant PTen with high-level B7-H1 expression37. The

acquisition of lysis resistance by tumour cells occurs rapidly, within 4–16 hours, and was also observed for allogeneic27,105 and re-directed T cells37 in addition to TAA-specific T cells. This effect could be interpreted in terms of the rapid induction of suppressive effects on T cells through interaction with PD-1. However, further stud-ies showed that this was due to the unique nature of the tumour-cell surface, but not to the dysfunction of T cells, because wild-type tumour cells in the presence of B7-H1+ tumour cells in the same culture could still be lysed by T cells82. By disabling the intracellular domain of B7-H1 on cancer cells, the ‘molecular shield’ is eliminated and the tumour cells become susceptible in vitro to immune- mediated destruction. Importantly, tumours expressing B7-H1 with a truncated intracellular domain also become more susceptible to T-cell-mediated immunotherapy in vivo compared with tumours expressing wild-type B7-H1. By contrast, the intracellular truncation of PD-1 on T cells does not eliminate the ‘molecular-shield’ effect27. In addition, B7-H1 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 expres-sion pattern, B7-H1 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.

IL‑10 production. Selective induction of IL-10 was found initially following the stimulation of human T cells with CD3-specific antibody and B7-H1–immunoglobulin fusion protein20. Tumour-associated B7-H1+ DCs also induce T-cell production of IL-10 (ReF. 23). Subsequent studies have established a correlation between the upreg-ulation of B7-H1 expression and increased levels of IL-10 in patients with HIv and HBv infection107,108. It remains to be determined whether increased IL-10 production has an important role in B7-H1-mediated suppression of T-cell responses in vivo.

TReg‑cell‑mediated suppression. It has been suggested that B7-H1 is involved in the development and function of TReg cells. B7-H1+ vascular endothelial cells109 and gastric epithelial cells110 induce the development of TReg cells in an in vitro culture system. However, it has not been shown that B7-H1 or PD-1 is required for TReg-cell differentia-tion in vivo. In tumour-bearing mice, TReg cells activated by IDO+ DCs induce B7-H1 expression on target DCs. Importantly, the ability of these TReg cells to suppress T-cell activation could be abrogated by antibody specific for B7-H1 (ReF. 111). In support of this observation, it has been shown that human tumour-associated DCs express B7-H1 and mediate T-cell suppression in a B7-H1-dependent manner23,45. By contrast, in non-Hodgkin lym-phoma, intratumoral CD4+CD25+ TReg cells can express B7-H1, and blocking B7-H1 with a monoclonal antibody partially decreases TReg-cell-mediated T-cell inhibition112. These results indicate that B7-H1 expression by tumour-associated DCs and TReg cells might contribute to tumour immune suppression.

Figure 3 |TheinhibitoryactionsofB7‑H1intumourimmuneevasion.Tumour cells and tumour-associated antigen-presenting cells (APCs) express high levels of B7-H1. The potential suppressive mechanisms of B7-H1 have been addressed in various models in vitro and in vivo, and it is probable that a combination of mechanisms, rather than a single mode of action, is used by B7-H1-expressing cells. B7-H1+ tumour cells and APCs might induce T-cell apoptosis, anergy, functional exhaustion or IL-10 production. B7-H1+ 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 B7-H1 and mediate T-cell suppression partially through the B7-H1–PD-1 pathway. The detailed molecular mechanisms remain poorly understood.

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Small interfering RNADouble-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 plasmid-delivered short hairpin RNAs (shRNAs).

Antisense oligonucleotidesShort, 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.

B7‑H4 can also evade tumour immunity. B7-H4 has been studied in less detail than B7-H1 in the context of tumour immune evasion, but there is evidence indicat-ing that B7-H4 might exert its function through myeloid APCs and TReg cells to mediate T-cell suppression in the tumour microenvironment31,55,74. Tumour-associated macrophages markedly outnumber other types of APC, such as DCs, and form an abundant population of APCs in solid tumours113–116. One population of ovarian-cancer-associated macrophages expresses high levels of B7-H4, and these B7-H4+ macrophages induce T-cell cycle arrest in vitro and in vivo partially through B7-H4 (ReF. 31). These findings indicate that B7-H4 might contribute to tumour immune evasion, and that B7-H4 is a molecular target for tumour immunotherapy.

In human ovarian cancer, TReg cells and B7-H4+ macrophages are co-localized and their numbers are correlated in the tumour environment16,31,55,74. TReg cells trigger high levels of IL-6 and IL-10 production by APCs, and in turn, these cytokines stimulate the expression of B7-H4 by APCs, which renders the APCs immuno-suppressive55 (FIG. 2). These findings are in line with the observations that macrophages spontaneously produce IL-6 and IL-10 in the ovarian tumour environment55,62. These data mechanistically link IL-10, B7-H4, 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 immunotherapyMany tumour-associated APCs and tumour cells express B7-H1 and B7-H4, and these molecules can mediate T-cell suppression. The manipulation of B7-induced immune suppression might therefore be a broadly appli-cable therapeutic modality to treat human cancers.

B7‑H1 and PD‑1 blockade. Preclinical data have set the stage for clinical trials by blocking B7-H1 in patients with cancer. In the context of its suppressive effects on T cells and anti-apoptotic effect on tumour cells, it would seem beneficial to block B7-H1 using neutralizing antibodies. However, antibodies specific for B7-H1 could potentially also block the interaction of B7-H1 with a putative co-stimulatory receptor. In this regard, it has been reported that local expression of B7-H1 can provide positive co-stimulation for naive T cells and promote organ-specific autoimmunity and transplant rejection in mice117. For obvious reasons, therapeutic antibodies should be tested for their ability to block the B7-H1–CD80 interaction in addition to blocking B7-H1–PD-1 binding. Despite the possible immune-stimulatory roles of B7-H1 in some mouse models of transplantation and autoimmune diseases, the effects of B7-H1 are largely immunosup-pressive on mouse and human tumour immunity. In terms of the suppressive effects of B7-H1, blocking B7-H1 could possibly enhance ongoing autoimmune diseases. For example, treatment with B7-H1-specific antibody moderately accelerates experimental autoim-mune encephalomyelitis in mice118. B7-H1-deficient nOD mice also develop diabetes more rapidly than wild-type nOD mice119, and the loss of B7-H1 expression on

non-haematopoietic cells is crucial for the accelerated disease120. However, in light of the mild autoimmune phenotypes in B7-H1-deficient mice75,76, B7-H1 block-ade will be unlikely to result in the generation of severe autoimmune disease.

In addition to the effects of B7-H1 on transplantation, autoimmune diseases and tumours, it has been found that trophoblasts, which are located in the maternal–fetal interface, express B7-H1, and B7-H1+ trophoblasts might have a role in the suppression of maternal–fetal immunity42. Therefore, a potential side effect of B7-H1 blockade by antibody in pregnant women could be the termination of pregnancy. nevertheless, B7-H1-deficient mice produce normal size litters, which indicates that this is also a less likely event.

Similarly, neutralizing antibodies specific for PD-1 will be an important addition to clinical trials. However, neutralizing antibodies specific for PD-1 might block the interaction of PD-1 with B7-DC (FIG. 1), another counter-receptor for PD-1 with potentially stimulatory effects. In addition, PD-1-specific antibodies might increase the risk of severe autoimmune diseases, as predicted from the spontaneous development of autoimmune diseases in PD-1-deficient mice17,18. Finally, although in vitro assays support the claim that certain specific monoclonal antibodies are antagonistic, if appropriate crosslinking is provided, these antibodies could be potentially agonis-tic121. nonetheless, the significant potential benefits of the clinical application of B7-H1–PD-1 blockade warrant extensive experimental and clinical studies in patients with cancer.

B7‑H4 blockade. efficient neutralizing antibodies specific for human B7-H4 are not yet available. Small interfering RNA (siRnA)54 and antisense oligonucleotides specific for B7-H4 (ReFS 31,74) have been used to block B7-H4 expression. Blocking the expression of B7-H4 by tumour-associated macrophages disables their suppressive capacity, enables TAA-specific effector T-cell function and reduces tumour growth in human ovarian cancer xenografts31,74. Taken together with the proposed inhibitory role for B7-H4 in mouse systems28–30, these data justify B7-H4 as a promis-ing 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 T-cell immunity. In tumour-bearing mouse models, for example, blocking B7-H1 in combination with blocking TGFβ signalling using neutralizing antibodies synergistically induces tumour regression133. B7-H1 expression is induced on tumour-associated TReg cells in non-Hodgkin lym-phoma and on target DCs in murine tumour-draining lymph nodes. These TReg cells inhibit the function of PD-1+ tumour-infiltrating T cells, partially through the B7-H1–PD-1 pathway111,112. These studies indicate that simultaneously blocking B7-H1–PD-1 and TReg cells might be therapeutically relevant.

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The administration of a blocking monoclonal anti-body specific for B7-H1 increases the therapeutic effects of a co-stimulatory CD137-specific agonistic monoclonal antibody82,122 and of tumour-cell vaccination123. B7-H1 and PD-1 blockade together with an HSP70 vaccine124 can increase T-cell immunity and decrease tumour growth and metastasis in mouse models. In addition to these examples, blocking the B7-H1–PD-1 pathway in combination with blockade of other well-defined sup-pressive molecules, including CTLA4, veGF, B7-H4, TGFβ, arginase or IDO1, could be possible options in preclinical and clinical settings to treat cancer.

Concluding remarksThere is now compelling evidence to show that tumours escape host immunity by actively developing multiple suppressive mechanisms in the tumour microenviron-ment. The mechanisms that underlie the interactions between the immune system and the tumour micro-environment, particularly in humans, are a crucial and understudied area of cancer immunology, the under-standing 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 impor-tant immunosuppressive mechanism in many types of human tumour. Therefore, the manipulation of the expression of and signalling through these molecules is

an attractive strategy to treat human cancers. However, as we move forward to clinical trials, we have to bear in mind several possibilities. First, the immune suppres-sion mediated by inhibitory B7 molecules might not be the only or the main immunosuppressive mechanism used by certain tumour stages and/or certain types of tumour. Therefore, it is essential to define the nature and functional relevance of inhibitory B7-family members in each individual human tumour microenvironment. Second, the interactions between the B7-family mem-bers and their receptors are complex and might reflect differences in temporal and spatial expression of these molecules and their affinities for one another in the tumour microenvironment. Third, blocking inhibitory B7 molecules might result in autoimmune diseases. Finally, the receptor(s) and signalling pathways have not been identified for several B7-family members. These limitations will probably constrain the clinical applica-tion of targeting inhibitory B7-family members as a therapeutic modality. Identification of all the counter-receptors in these pathways and careful analysis of their positive and negative regulatory functions will help us to design more elegant strategies to maximize anti-tumour immunity while decreasing the risk of autoimmune dis-eases. In addition, targeting one pathway will be highly unlikely to lead to reliable and consistent clinical effi-cacy, and combinatorial therapeutic regimens will need to be developed.

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AcknowledgementsWe 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.

DATABASESEntrez Gene: http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=geneB7-DC | B7-H1 | B7-H2 | B7-H3 | B7-H4 | CD80 | CD86

FURTHER INFORMATIONWeiping Zou’s homepage: http://sitemaker.umich.edu/zou.lab/home Lieping Chen’s homepage: http://gradimmunology.med.som.jhmi.edu

AlllinksAReAcTiveinTHeonlinepdf

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http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=gene

http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=gene








http://sitemaker.umich.edu/zou.lab/home

http://gradimmunology.med.som.jhmi.edu

inhibitory b7 family molecules in tumour microenvironment

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