Ovarian cancer is the most lethal of all gynecological cancers, and there is an urgent unmet need to develop new therapies. Epithelial ovarian cancer (EOC) is characterized by an immune suppressive microenvironment, and response of ovarian cancers to immune therapies has thus far been disappointing. We now find, in a mouse model of EOC, that clinically relevant doses of DNA methyltransferase and histone deacetylase inhibitors (DNMTi and HDACi, respectively) reduce the immune suppressive microenvironment through type I IFN signaling and improve response to immune checkpoint therapy. These data indicate that the type I IFN response is required for effective in vivo antitumorigenic actions of the DNMTi 5-azacytidine (AZA). Through type I IFN signaling, AZA increases the numbers of CD45 immune cells and the percentage of active CD8 T and natural killer (NK) cells in the tumor microenvironment, while reducing tumor burden and extending survival. AZA also increases viral defense gene expression in both tumor and immune cells, and reduces the percentage of macrophages and myeloid-derived suppressor cells in the tumor microenvironment. The addition of an HDACi to AZA enhances the modulation of the immune microenvironment, specifically increasing T and NK cell activation and reducing macrophages over AZA treatment alone, while further increasing the survival of the mice. Finally, a triple combination of DNMTi/HDACi plus the immune checkpoint inhibitor α-PD-1 provides the best antitumor effect and longest overall survival, and may be an attractive candidate for future clinical trials in ovarian cancer.

No abstract

Efficacious antitumor immune responses must overcome multiple suppressive mechanisms in the tumor microenvironment to control cancer progression. In this study, we demonstrate that dual targeting of suppressive myeloid populations by inhibiting CSF-1/CSF-1R signaling and activation of antigen-presenting cells with agonist anti-CD40 treatment confers superior antitumor efficacy and increased survival compared with monotherapy treatment in preclinical tumor models. Concurrent CSF-1R blockade and CD40 agonism lead to profound changes in the composition of immune infiltrates, causing an overall decrease in immunosuppressive cells and a shift toward a more inflammatory milieu. Anti-CD40/anti-CSF-1R-treated tumors contain decreased tumor-associated macrophages and Foxp3 þ regulatory T cells. This combination approach increases maturation and differentiation of proinflammatory macrophages and dendritic cells and also drives potent priming of effector T cells in draining lymph nodes. As a result, tumor-infiltrating effector T cells exhibit improved responses to tumor antigen rechallenge. These studies show that combining therapeutic approaches may simultaneously remove inhibitory immune populations and sustain endogenous antitumor immune responses to successfully impair cancer progression.

Although ovarian cancer has a low incidence rate, it remains the most deadly gynecologic malignancy. Previous work has demonstrated that the DNMTi 5-Azacytidine (5AZA-C) activates type I interferon signaling to increase IFNg þ T cells and natural killer (NK) cells and reduce the percentage of macrophages in the tumor microenvironment. To improve the efficacy of epigenetic therapy, we hypothesized that the addition of a-difluoromethylornithine (DFMO), an ornithine decarboxylase inhibitor, may further decrease immunosuppressive cell populations improving outcome. We tested this hypothesis in an immunocompetent mouse model for ovarian cancer and found that in vivo, 5AZA-C and DFMO, either alone or in combination, significantly increased survival, decreased tumor burden, and caused recruitment of activated (IFNg þ) CD4 þ T cells, CD8 þ T cells, and NK cells. The combination therapy had a striking increase in survival when compared with single-agent treatment , despite a smaller difference in recruited lymphocytes. Instead, combination therapy led to a significant decrease in immunosuppressive cells such as M2 polarized macrophages and an increase in tumor-killing M1 macrophages. In this model, depletion of macrophages with a CSF1Rblocking antibody reduced the efficacy of 5AZA-C þ DFMO treatment and resulted in fewer M1 macrophages in the tumor microenvironment. These observations suggest our novel combination therapy modifies macrophage polarization in the tumor microenvironment, recruiting M1 macrophages and prolonging survival. Significance: Combined epigenetic and polyaminereducing therapy stimulates M1 macrophage polarization in the tumor microenvironment of an ovarian cancer mouse model, resulting in decreased tumor burden and prolonged survival.

IntroductionProtection against recurrent infections resulting from the same pathogen is a hallmark of adaptive immunity. After acute infection by an intracellular pathogen, naive CD8 ϩ T cells expressing epitope-specific T-cell receptors (TCRs) are activated. The effector phase of the response is short, with a rapid expansion of antigenspecific T cells and pathogen clearance. The expanded effector cells undergo a contraction phase, while approximately 5% to 10% of antigen-specific cells are maintained to establish a memory pool and provide long-term protection from reinfection by the same pathogen. [1][2][3] In the early stages of the effector response to acute lymphocytic choriomeningitis virus (LCMV) infection, activated CD8 ϩ cells differentiate into 2 subsets with distinct fates. These populations can be phenotypically identified by cell-surface expression of killer cell lectin-like receptor G1 (KLRG-1) and the receptor for interleukin-7 (IL-7R). 4 Short-lived effector cells (SLECs) express high levels of KLRG-1 and the transcription factors Blimp-1 and T-bet, and decreased levels of IL-7R. [5][6][7][8] SLECs are dependent on signals from the environment, including TCR signals, inflammatory cytokines such as IL-12 and IFN␥, and common ␥ chain cytokine signaling from IL-2 and IL-15. 4,9 Conversely, memory precursor (MP) cells express low levels of KLRG-1 and higher levels of IL-7R␣, CXCR3, and CD27. 4,10,11 While these cells possess effector function, they also have the potential to further differentiate into long-lived memory T cells after the resolution of infection. The molecular nature of the proximal signals involved in the SLEC/MP cell-fate decision and those required for normal homeostasis of these populations have not been extensively studied.Previous studies have shown that IL-15-and IL-7-generated signals are required for memory T-cell homeostasis. [12][13][14][15] Depending on the experimental system and the characteristics used to define the memory population, TCR signals have been shown to be required or dispensable. For example, H-2D b -restricted, malespecific (H-Y TCR-transgenic) memory CD8 ϩ T cells require expression of either H-2D b or H-2D d for survival. 16 The absence of all major histocompatibility complex (MHC) class I expression leads to the disappearance of the cells, suggesting that a tonic MHC-TCR signal is required. In addition, CD8 ϩ CD44 hi cells do not persist in mice after gene deletion of the TCR␣ chain. 17 In contrast, polyclonal CD8 ϩ T-cell populations containing memory CD8 ϩ T cells generated by viral infection persist indefinitely when transferred into MHC class I-deficient mice. 18 CD44 hi cells and TCR-transgenic memory T cells persist long term, even when the expression of the src family tyrosine kinase Lck or of TCR itself is substantially decreased by a bitransgenic tetracycline regulatory system. [19][20][21] An obstacle to characterizing the requirements for memory population generation and persistence is the heterogeneity of definitions for memory CD8 ϩ T cells o...

Dendritic cells (DC) are important regulators of T cell immunity. The degree of stimulation, the pattern of costimulatory molecules expressed, and the cytokines secreted by DC dictate the nature of the effector and memory cells generated, particularly with respect to their Th1 or Th2 phenotypes. In this study, we demonstrate that the addition of activated DC to spleen cultures containing established Th2-polarized CD4+ T cells was sufficient to suppress Th2 and induce Th1 cytokines in a recall response, a phenomenon referred to as phenotype reversal. The ability of activated DC to induce phenotype reversal displayed exquisite Ag specificity. The DC activator B7-DC cross-linking Ab (XAb) was >10,000-fold more efficient at inducing phenotype reversal than the TLR agonists CpG-oligodeoxynucleotide and Gardiquimod. Characterization of the mechanisms governing phenotype reversal revealed the requirement for cognate interaction between the TCR:peptide-MHC complex, the expression of the costimulation/adhesion molecule ICAM-1, and secretion of IL-12 and IFN-γ by the activated DC. The requirement for the costimulation/adhesion molecule SLAM (signaling lymphocytic activation molecule) was found to be quantitative. Thus, activation of DC, particularly by crosslinking B7-DC, can modulate well-established Th2 T cell responses in an Ag-specific manner. Because the regulation of mouse and human DC by B7-DC XAb overlaps in several significant ways, immune modulation with B7-DC XAb is a potential strategy for treating Th2-mediated diseases.

Transcription factors regulate T cell fates at every stage of development and differentiation. Members of the Foxp family of forkhead transcription factors are essential for normal T lineage development; Foxp3 is required for T regulatory cell generation and function, and Foxp1 is necessary for generation and maintenance of naïve T cells. Foxp4, an additional member of the Foxp family, is highly homologous to Foxp1 and has been shown to dimerize with other Foxp proteins. We report the initial characterization of Foxp4 in T lymphocytes. Foxp4 is expressed in both thymocytes and peripheral CD4+ and CD8+ T cells. We used a CD4Cre mediated approach to evaluate the cell autonomous role for Foxp4 in murine T lymphocytes. T cell development, peripheral cellularity and cell surface phenotype are normal in the absence of Foxp4. Furthermore, Foxp3+ T regulatory cells develop normally in Foxp4 deficient animals and naïve Foxp4 deficient CD4 T cells can differentiate to inducible T regulatory cells in vitro. In wild-type T cells, expression of Foxp4 increases following activation, but deletion of Foxp4 does not affect T cell proliferative responses or in vitro effector T cell differentiation. In vivo, despite effective control of Toxoplasma gondii and acute lymphocytic choriomeningitis virus infections, effector cytokine production during antigen specific recall responses are reduced in the absence of Foxp4. We conclude that Foxp4 is dispensable for T cell development, but necessary for normal T cell cytokine recall responses to antigen following pathogenic infection.

No abstract