Manipulating the Wnt/ß-catenin signaling pathway to promote anti-tumor immune infiltration into the TME to sensitize ovarian cancer to ICB therapy.

OBJECTIVE: Immune checkpoint blockade (ICB) therapy shows limited efficacy in ovarian cancers due to the "cold" immune phenotype surrounding these tumors. Previous studies have shown that in ovarian cancer Wnt/ß-catenin pathway activation contributes to this immune phenotype. Here, we evaluated the anti-tumor and immune-enhancing properties of the Wnt inhibitor, CGX-1321, used alone or in combination with either DKN-01 or anti-PD-1 therapy, in pre-clinical ovarian cancer models. METHODS: The parental ID8 murine ovarian cancer model harboring a knock-out of p53 (ID8p53-/-) and MISIIR-Tag spontaneous ovarian cancer models were used to test the effects of CGX-1321 alone or in combination therapies on tumor burden and immune cell landscape in the tumor microenvironment (TME). Flow cytometry and NanoString analyses were used to characterize the changes in tumor-intrinsic signaling and immune-related profiles in the TME of ovarian cancer in response to treatments. RESULTS: CGX-1321 significantly reduced tumor burden and constrained tumor progression in the ID8p53-/- and MISIIR-Tag models. Furthermore, CGX-1321 increased infiltrating CD8+ T cells in the TME. Combining CGX-1321 with either DKN-01 or anti-PD-1 therapy also decreased tumor burden and increased CD8+ T cell infiltration in the omentum TME but did not do so to a greater extent that CGX-1321 monotherapy. CONCLUSIONS: CGX-1321 significantly reduced tumor burden and enhanced CD8+ T cell levels in ovarian cancer, nevertheless the addition of DKN-01 or anti-PD-1 therapies did not enhance these effects of CGX-1321. Further investigation is needed to determine if CGX-1321 + DKN-01 combination treatment sensitizes pre-clinical ovarian cancer to ICB therapy.

Mycoplasma bovoculi--augmented bovine natural killer activity.

The effect of conjunctival Mycoplasma bovoculi infection on local and systemic natural killer activity was studied in cattle. A standard in vitro assay against the mouse tumor cell line, YAC-1, was used to measure increases in effector cell activity. Local responses in the eye were investigated by examination of stained exfoliative conjunctival cells and tissue biopsies. An increase in systemic cytolytic activity was seen in infected calves, and maximal values were observed on the second day post infection (DPI). An elevation in the number of large granular lymphocytes was noted in the conjunctival samples, and maximal numbers were again observed at 2 days post infection. It is proposed that natural killer cell activity may play a role in the initial host responses of cattle to M. bovoculi infection.

Intracellular adapter molecules.

Lymphocyte antigen receptor engagement leads to the initiation of numerous signal transduction pathways that direct ultimate cellular responses. In recent years, it has become apparent that adapter molecules regulate the coupling of receptor-proximal events, such as protein tyrosine kinase activation, with end results such as inducible gene expression and cytoskeletal rearrangements. While adapter molecules possess no intrinsic enzymatic activity, their ability to mediate protein-protein interactions is vital for the integration and propagation of signal transduction cascades in lymphocytes. Recent studies demonstrate that intracellular adapter molecules function as both positive and negative regulators of lymphocyte activation.

A newly identified response element in the CD95 ligand promoter contributes to optimal inducibility in activated T lymphocytes.

Inducible expression of CD95 ligand on activated T lymphocytes contributes to both cytotoxic effector mechanisms and peripheral T cell homeostasis. To understand better the transcriptional events that regulate this expression, we have examined the CD95 ligand promoter to determine which regions are required for its induced activity following T cell stimulation. We report here the identification of a new response element within the promoter that is required for its optimal function in activated Jurkat T cells. This region is bound by proteins contained in nuclear extracts of activated, but not resting, T cells. Multimerization of this sequence independently drives transcription in response to T cell activation, while mutation of it substantially decreases inducible promoter activity. Finally, we provide evidence that T cell activation-induced transcription of the CD95 ligand gene is regulated coordinately by this response element together with two previously defined sites for nuclear factor of activated T cells (NFAT).

Two NFAT transcription factor binding sites participate in the regulation of CD95 (Fas) ligand expression in activated human T cells.

Antigen receptor engagement on T lymphocytes activates transcription factors important for stimulating cytokine gene expression. This is critical for clonal expansion of antigen-specific T cells and propagation of immune responses. Additionally, under some conditions antigen receptor stimulation initiates apoptosis of T lymphocytes through the induced expression of CD95 ligand and its receptor. Here we demonstrate that the transcription factor, NFAT, which is critical for the inducible expression of many cytokine genes, also plays a critical role in the regulation of T cell receptor-mediated CD95 ligand expression. Two sites within the CD95 ligand promoter, identified through DNase I footprinting, bind NFAT proteins from nuclear extracts of activated T cells. Although both sites appear important for optimal expression of CD95 ligand in activated T cells, mutational analysis suggests that the distal NFAT site plays a more significant role. Furthermore, these sites do not appear to be required for constitutive CD95 ligand expression in Sertoli cells.

Regulation of CD95 (Fas) ligand expression by TCR-mediated signaling events.

Stimulation of mature peripheral T cells by TCR engagement results in activation of signals that drive induction of cytokine gene expression and clonal expansion. However, under some conditions, engagement of the TCR leads instead to apoptosis. Recent studies demonstrate that TCR-stimulated apoptosis requires expression of CD95 ligand on activated T cells followed by an interaction between CD95 ligand and the CD95 receptor also expressed on this population. The experiments reported in this study were designed to address the signaling events triggered by TCR engagement that are important for regulating CD95 ligand gene expression. To approach this, we generated a luciferase reporter construct containing elements of the CD95 ligand promoter. Using a previously described mutant of the Jurkat T cell line, we show that proximal signaling events dependent on the presence of the CD45 tyrosine phosphatase are required for TCR-stimulated CD95 ligand expression. Transient transfection studies demonstrate further that TCR-stimulated activation of the Ras signaling pathway is required for optimal activation of CD95 ligand. Next, in an effort to determine critical transcription factors that regulate CD95 ligand expression, we demonstrate a cyclosporin A-sensitive nuclear factor-AT response element in the promoter region of this gene that is critical for optimal CD95 ligand reporter activity in stimulated T cells. Together, these studies begin a dissection of the biochemical events that lead to expression of CD95 ligand, a required step for TCR-induced apoptosis.

The regulation of CD95 (Fas) ligand expression in primary T cells: induction of promoter activation in CD95LP-Luc transgenic mice.

The interaction between CD95 (Fas) and CD95L (Fas ligand) initiates apoptosis in a variety of cell types. Although the regulation of CD95L expression on activated T cells is an area of intense study, knowledge related to the induction of CD95L promoter activity in primary T cells is lacking. In this report we describe the generation of a novel transgenic mouse strain, CD95LP-Luc, in which murine CD95L promoter sequence controls the expression of a luciferase reporter gene. We use these mice to illustrate several important findings related to transcriptional regulation of CD95L in primary T cells. We demonstrate that maximal CD95L promoter activity occurs only after prolonged T cell stimulation and requires costimulation through CD28. We provide evidence that thymocytes express CD95L/luciferase after strong TCR ligation and that inducible CD95L promoter activation is present, but unequal, in both Th1 and Th2 effector cells. We also illustrate that while agonist peptide presentation by APCs generates robust proliferation during a primary T cell response, the same stimulus induces only modest CD95L promoter activity. These results suggest alternate explanations for the well-characterized delay in CD95-mediated activation-induced cell death following initial ligation of the TCR.

Differential regulation of the IL-12 p40 promoter and of p40 secretion by CpG DNA and lipopolysaccharide.

Challenge of macrophages with DNA containing an internal CpG motif results in IL-12 p40 secretion. In the presence of IFN-gamma, CpG DNA induces more p40 secretion than does LPS. In the RAW 264 macrophage cell line, both CpG DNA and LPS activate a p40 promoter-reporter construct, and the promoter response to either agent is augmented 2- to 5-fold by IFN-gamma. While either LPS or CpG DNA induces p40 promoter activity, only CpG DNA induces an increase in p40 mRNA or protein secretion. Even though IFN-gamma augmented LPS-driven p40 promoter activity in RAW 264 cells, the combination of IFN-gamma and LPS induced less p40 mRNA or protein secretion than the combination of IFN-gamma and CpG DNA. The ability of IFN-gamma to augment LPS or CpG DNA-induced p40 promoter activation was observed with truncation mutants of the IL-12 promoter containing as few as 250 bp 5' of the TATA box. Although LPS alone is a poor inducer of p40 transcription, both LPS and CpG DNA induce similar nuclear translocation of NF-kappaB. This binding is not augmented by costimulation with IFN-gamma. Thus, CpG DNA induces p40 transcription by a mechanism that includes NF-kappaB translocation; however, CpG DNA appears to induce other factor(s) necessary for p40 transcription. These results illustrate fundamental differences between CpG DNA and LPS with respect to activation of IL-12 p40 secretion.