Programmed death 1 (PD-1) is a new member of the CD28/CTLA-4 family, which has been implicated in the maintenance of peripheral tolerance. Two ligands for PD-1, namely, B7-H1 (PD-L1) and B7-DC (PD-L2), have recently been identified as new members of the B7 family but their expression at the protein level remains largely unknown. To characterize the expression of B7-H1 and B7-DC, we newly generated an anti-mouse B7-H1 mAb (MIH6) and an anti-mouse B7-DC mAb (TY25). MIH6 and TY25 immunoprecipitated a single molecule of 43 and 42 kDa from the lysate of B7-H1 and B7-DC transfectants, respectively. Flow cytometric analysis revealed that B7-H1 was broadly expressed on the surface of mouse tumor cell lines while the expression of B7-DC was rather restricted. PD-1 was expressed on anti-CD3-stimulated T cells and anti-IgM plus anti-CD40-stimulated B cells at high levels but was undetectable on activated macrophages or DCs. B7-H1 was constitutively expressed on freshly isolated splenic T cells, B cells, macrophages, and dendritic cells (DCs), and up-regulated on T cells by anti-CD3 stimulation on macrophages by LPS, IFN-γ, GM-CSF, or IL-4, and on DCs by IFN-γ, GM-CSF, or IL-4. In contrast, B7-DC expression was only inducible on macrophages and DCs upon stimulation with IFN-γ, GM-CSF, or IL-4. The inducible expression of PD-1 ligands on both T cells and APCs may suggest new paradigms of PD-1-mediated immune regulation.

Programmed death-1 (PD-1) receptor, an inhibitory costimulatory molecule found on activated T cells, has been demonstrated to play a role in the regulation of immune responses and peripheral tolerance. We investigated the role of this pathway in the development of autoimmune diabetes. PD-1 or PD-L1 but not PD-L2 blockade rapidly precipitated diabetes in prediabetic female nonobese diabetic (NOD) mice regardless of age (from 1 to 10-wk-old), although it was most pronounced in the older mice. By contrast, cytotoxic T lymphocyte–associated antigen 4 (CTLA-4) blockade induced disease only in neonates. Male NOD mice also developed diabetes after PD-1–PD-L1 pathway blockade, but NOR mice, congenic to NOD but resistant to the development of diabetes, did not. Insulitis scores were significantly higher and frequency of interferon γ–producing GAD-reactive splenocytes was increased after PD-1–PD-L1 pathway blockade compared with controls. Interestingly, PD-L1 but not PD-L2 was found to be expressed on inflamed islets of NOD mice. These data demonstrate a central role for PD-1–PD-L1 interaction in the regulation of induction and progression of autoimmune diabetes in the NOD mouse and provide the rationale to develop new therapies to target this costimulatory pathway in this disease.

The mechanisms by which tumor microenvironments modulate nucleic acid–mediated innate immunity remain unknown. Here we identify the receptor TIM-3 as key in circumventing the stimulatory effects of nucleic acids in tumor immunity. Tumor-associated dendritic cells (DCs) in mouse tumors and patients with cancer had high expression of TIM-3. DC-derived TIM-3 suppressed innate immune responses through the recognition of nucleic acids by Toll-like receptors and cytosolic sensors via a galectin-9-independent mechanism. In contrast, TIM-3 interacted with the alarmin HMGB1 to interfere with the recruitment of nucleic acids into DC endosomes and attenuated the therapeutic efficacy of DNA vaccination and chemotherapy by diminishing the immunogenicity of nucleic acids released from dying tumor cells. Our findings define a mechanism whereby tumor microenvironments suppress antitumor immunity mediated by nucleic acids.

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Experimental autoimmune encephalomyelitis (EAE) is mediated by autoantigen-specific T cells dependent on critical costimulatory signals for their full activation and regulation. We report that the programmed death-1 (PD-1) costimulatory pathway plays a critical role in regulating peripheral tolerance in murine EAE and appears to be a major contributor to the resistance of disease induction in CD28-deficient mice. After immunization with myelin oligodendrocyte glycoprotein (MOG) there was a progressive increase in expression of PD-1 and its ligand PD-L1 but not PD-L2 within the central nervous system (CNS) of mice with EAE, peaking after 3 wk. In both wild-type (WT) and CD28-deficient mice, PD-1 blockade resulted in accelerated and more severe disease with increased CNS lymphocyte infiltration. Worsening of disease after PD-1 blockade was associated with a heightened autoimmune response to MOG, manifested by increased frequency of interferon γ–producing T cells, increased delayed-type hypersensitivity responses, and higher serum levels of anti-MOG antibody. In vivo blockade of PD-1 resulted in increased antigen-specific T cell expansion, activation, and cytokine production. Interestingly, PD-L2 but not PD-L1 blockade in WT animals also resulted in disease augmentation. Our data are the first demonstration that the PD-1 pathway plays a critical role in regulating EAE.

Strategies to activate and rescue exhausted tumor-specific T cells, including the use of monoclonal antibodies (mAb) that block the negative costimulatory receptors CTLA-4 and PD-1 are proving very effective, but TIM3 has been relatively neglected as a target. Here we report an extensive characterization of the therapeutic activity and mechanism of action of an anti-mouse TIM3 mAb against experimental and carcinogen-induced tumors. For the first time we specifically define the mechanism of antitumor action of anti-TIM3 requiring IFN-g producing CD8 þ T cells and CD4 þ T cells, and a higher ratio of tumor infiltrating CD8 þ

IntroductionApoptosis is a crucial process in the development and homeostasis of multicellular organisms. 1,2 In the immune system, an enormous number of cells undergo apoptosis during development of lymphocytes and after interaction with antigens. 3 Because apoptotic cells and secondary necrotic cells releasing intracellular contents could be autoantigens, phagocytes such as macrophages and dendritic cells (DCs) must engulf these dying cells rapidly and efficiently to prevent detrimental inflammatory responses and autoimmunity. 1,4 To engulf apoptotic cells, macrophages use a variety of molecules, including Mer tyrosine kinase (MerTK), 5 milk fat globule-EGF-factor 8 (MFG-E8), 6 brainspecific angiogenesis inhibitor 1 (BAI1), 7 and T-cell immunoglobulin and mucin domain-containing molecule 4 (Tim-4). 8,9 However, their relative contributions to the phagocytosis remain to be elucidated. Multiple receptors may simultaneously recognize multiple "eat-me" signals on apoptotic cells. In addition, different subsets of macrophages may use different repertoires of receptors for the phagocytosis.DCs are able to not only phagocytose apoptotic cells but also present dying cell-associated antigens with MHC class I molecules, which is termed as "cross-presentation." 1,10 It has been considered that, in steady state, cross-presentation of selfantigens by DCs stimulates CD8 ϩ T cells to proliferate abortively, resulting in their deletion, which is crucial to maintain peripheral tolerance. [10][11][12][13][14] Among mouse splenic DC subsets, CD8 ϩ DCs are unique in their ability for efficient phagocytosis of apoptotic cells and cross-presentation. 15,16 However, the mechanism for the recognition of apoptotic cells by CD8 ϩ DCs is poorly understood. Scavenger receptor CD36 and mannose receptor (MR)/DEC205 are highly expressed on CD8 ϩ DCs, but not CD8 Ϫ DCs, however, these receptors are not required for cross-presentation of cell-associated antigens by this DC subset. [16][17][18] Neither ␣ v ␤ 3 nor ␣ v ␤ 5 integrin that mediates phagocytosis of apoptotic cells by macrophages 1 is essential for phagocytosis by CD8 ϩ DCs. 17 Thus, the phagocytic receptor for apoptotic cells linked to cross-presentation remains to be identified.Tim-3 has been identified as a Th1-specific marker, and several in vivo studies have shown that Tim-3 regulates autoimmunity. 19,20 We and others have reported that Tim-3 negatively regulates Th1-mediated inflammatory diseases such as experimental autoimmune encephalomyelitis (EAE), type I diabetes, and acute graftversus-host diseases (aGVHD). [21][22][23] Moreover, it has been reported that Tim-3 promotes tolerance induction. 21,22 Recently, Zhu et al have identified galectin-9 as a Tim-3 ligand, and they have demonstrated that galectin-9 binds to the carbohydrate chains on Tim-3, and induces cell death of Th1 cells in vitro, which may explain the mechanism by which Tim-3 suppresses Th1 immunity. 24 On the other hand, Anderson et al have reported that Tim-3 is expressed on DCs, and that galectin-9 activate...

Microfold cells (M cells) are specialized epithelial cells situated over Peyer’s patches (PP) and other organized mucosal lymphoid tissues that transport commensal bacteria and other particulate Ags into intraepithelial pockets accessed by APCs. The TNF superfamily member receptor activator of NF-κB ligand (RANKL) is selectively expressed by subepithelial stromal cells in PP domes. We found that RANKL null mice have <2% of wild-type levels of PP M cells and markedly diminished uptake of 200 nm diameter fluorescent beads. Ab-mediated neutralization of RANKL in adult wild-type mice also eliminated most PP M cells. The M cell deficit in RANKL null mice was corrected by systemic administration of exogenous RANKL. Treatment with RANKL also induced the differentiation of villous M cells on all small intestinal villi with the capacity for avid uptake of Salmonella and Yersinia organisms and fluorescent beads. The RANK receptor for RANKL is expressed by epithelial cells throughout the small intestine. We conclude that availability of RANKL is the critical factor controlling the differentiation of M cells from RANK-expressing intestinal epithelial precursor cells.