TIM-3 is constitutively expressed on subsets of macrophages and dendritic cells. Its expression on other cells of the innate immune system and its role in fetomaternal tolerance has not yet been explored. Here we investigate the role of TIM-3 expressing innate immune cells in the regulation of tolerance at the fetomaternal interface (FMI) using an allogeneic mouse model of pregnancy. Blockade of TIM-3 results in accumulation of inflammatory granulocytes and macrophages at the utero-placental interface and up regulation of pro-inflammatory cytokines. Furthermore, TIM-3 blockade inhibits the phagocytic potential of uterine macrophages resulting in a build up of apoptotic bodies at the utero-placental interface that elicits a local immune response. In response to inflammatory cytokines, Ly-6ChiGneg M-MDSCs (monocytic myeloid derived suppressor cells) expressing iNOS and arginase 1 are induced. However, these suppressive cells fail to down-regulate the inflammatory cascade induced by inflammatory granulocytes (Ly-6Cint Ghi) and apoptotic cells; the increased production of IFNγ and TNFα by inflammatory granulocytes leads to abrogation of tolerance at the fetomaternal interface and fetal rejection. These data highlight the interplay between cells of the innate immune system at the FMI and their influence on successful pregnancy in mice.

Covalent modification of histones is a fundamental mechanism of regulated gene expression in eukaryotes, and interpretation of histone modifications is an essential feature of epigenetic control. Bromodomains are specialized binding modules that interact with acetylated histones, linking chromatin recognition to gene transcription. Because of their ability to function in a domain-specific fashion, selective disruption of bromodomain:acetylated histone interactions with chemical probes serves as a powerful means for understanding biological processes regulated by these chromatin adaptors. Here we describe the discovery and characterization of potent and selective small molecule inhibitors for the bromodomains of CREBBP/EP300 that engage their target in cellular assays. We use these tools to demonstrate a critical role for CREBBP/EP300 bromodomains in regulatory T cell biology. Because regulatory T cell recruitment to tumors is a major mechanism of immune evasion by cancer cells, our data highlight the importance of CREBBP/ EP300 bromodomain inhibition as a novel, small moleculebased approach for cancer immunotherapy.

Prostaglandin E 2 (PGE 2), an arachidonic acid pathway metabolite produced by cyclooxygenase (COX)-1/2, has been shown to impair anti-tumor immunity through engagement with one or more E-type prostanoid receptors (EP 1-4). Specific targeting of EP receptors, as opposed to COX-1/2 inhibition, has been proposed to achieve preferential antagonism of PGE 2-mediated immune suppression. Here we describe the anti-tumor activity of MF-766, a potent and highly selective small-molecule inhibitor of the EP 4 receptor. EP 4 inhibition by MF-766 synergistically improved the efficacy of anti-programmed cell death protein 1 (PD-1) therapy in CT26 and EMT6 syngeneic tumor mouse models. Multiparameter flow cytometry analysis revealed that treatment with MF-766 promoted the infiltration of CD8 + T cells, natural killer (NK) cells and conventional dendritic cells (cDCs), induced M1-like macrophage reprogramming, and reduced granulocytic myeloidderived suppressor cells (MDSC) in the tumor microenvironment (TME). In vitro experiments demonstrated that MF-766 restored PGE 2-mediated inhibition of lipopolysaccharide (LPS)-induced tumor necrosis factor (TNF)-α production in THP-1 cells and human blood, and PGE 2-mediated inhibition of interleukin (IL)-2-induced interferon (IFN)-γ production in human NK cells. MF-766 reversed the inhibition of IFN-γ in CD8 + T-cells by PGE 2 and impaired suppression of CD8 + T-cells induced by myeloid-derived suppressor cells (MDSC)/PGE 2. In translational studies using primary human tumors, MF-766 enhanced anti-CD3-stimulated IFN-γ, IL-2, and TNF-α production in primary histoculture and synergized with pembrolizumab in a PGE 2 high TME. Our studies demonstrate that the combination of EP 4 blockade with anti-PD-1 therapy enhances antitumor activity by differentially modulating myeloid cell, NK cell, cDC and T-cell infiltration profiles.

In a successful pregnancy, the semiallogeneic fetus is not rejected by the maternal immune system, which implies tolerance mechanisms protecting fetal tissues from maternal immune attack. Here we report that the ICOS-B7h costimulatory pathway plays a critical role in maintaining the equilibrium at the fetomaternal interface. Blockade of this pathway increased fetal resorption and decreased fetal survival in an allogeneic pregnancy model (CBA female × B6 male). Locally in the placenta, levels of regulatory markers such as IDO and TGF-β1 were reduced after anti-B7h monoclonal antibody treatment, whereas levels of effector cytokines (eg, IFN-γ) were significantly increased. In secondary lymphoid organs, enhanced IFN-γ and granzyme B production (predominantly by CD8(+) T cells) was observed in the anti-B7h-treated group. The deleterious effect of B7h blockade in pregnancy was maintained only in CD4 knockout mice, not in CD8 knockout mice, which suggests a role for CD8(+) T cells in immune regulation by the ICOS-B7h pathway. In accord, regulatory CD8(+) T cells (in particular, CD8(+)CD103(+) cells) were significantly decreased after anti-B7h monoclonal antibody treatment, and adoptive transfer of this subset abrogated the deleterious effect of B7h blockade in fetomaternal tolerance. Taken together, these data support the hypothesis that B7h blockade abrogates tolerance at the fetomaternal interface by enhancing CD8(+) effector response and reducing local immunomodulation mediated by CD8(+) regulatory T cells.

NK cells are the most abundant lymphocyte population in the feto-maternal interface during gestation. The uterine NK cells (uNK) are transient, have a unique immunophenotype and produce a number of cytokines. These cytokines play an important role in establishment and maintenance of vascular remodeling and tolerance associated with successful pregnancy. The uNK cells also express TIM-3 during gestation and blockade of TIM-3 expression results in fetal loss in mice. In this study we determined the effect of TIM-3 blockade on uNK cells. Specifically we observed surface receptor phenotype and cytokine production by uNK cells following TIM-3 blockade. Our results show that TIM-3 plays a role in regulating the uNK cells and contributes to the maintenance of tolerance at the feto-maternal interface.

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