A SOX9-B7x axis safeguards dedifferentiated tumor cells from immune surveillance to drive breast cancer progression.

How dedifferentiated stem-like tumor cells evade immunosurveillance remains poorly understood. We show that the lineage-plasticity regulator SOX9, which is upregulated in dedifferentiated tumor cells, limits the number of infiltrating T lymphocytes in premalignant lesions of mouse basal-like breast cancer. SOX9-mediated immunosuppression is required for the progression of in situ tumors to invasive carcinoma. SOX9 induces the expression of immune checkpoint B7x/B7-H4 through STAT3 activation and direct transcriptional regulation. B7x is upregulated in dedifferentiated tumor cells and protects them from immunosurveillance. B7x also protects mammary gland regeneration in immunocompetent mice. In advanced tumors, B7x targeting inhibits tumor growth and overcomes resistance to anti-PD-L1 immunotherapy. In human breast cancer, SOX9 and B7x expression are correlated and associated with reduced CD8+ T cell infiltration. This study, using mouse models, cell lines, and patient samples, identifies a dedifferentiation-associated immunosuppression mechanism and demonstrates the therapeutic potential of targeting the SOX9-B7x pathway in basal-like breast cancer.

Recent advancements in the B7/CD28 immune checkpoint families: new biology and clinical therapeutic strategies.

The B7/CD28 families of immune checkpoints play vital roles in negatively or positively regulating immune cells in homeostasis and various diseases. Recent basic and clinical studies have revealed novel biology of the B7/CD28 families and new therapeutics for cancer therapy. In this review, we discuss the newly discovered KIR3DL3/TMIGD2/HHLA2 pathways, PD-1/PD-L1 and B7-H3 as metabolic regulators, the glycobiology of PD-1/PD-L1, B7x (B7-H4) and B7-H3, and the recently characterized PD-L1/B7-1 cis-interaction. We also cover the tumor-intrinsic and -extrinsic resistance mechanisms to current anti-PD-1/PD-L1 and anti-CTLA-4 immunotherapies in clinical settings. Finally, we review new immunotherapies targeting B7-H3, B7x, PD-1/PD-L1, and CTLA-4 in current clinical trials.

The immune checkpoint B7x expands tumor-infiltrating Tregs and promotes resistance to anti-CTLA-4 therapy.

Immune checkpoint molecules play critical roles in regulating the anti-tumor immune response, and tumor cells often exploit these pathways to inhibit and evade the immune system. The B7-family immune checkpoint B7x is widely expressed in a broad variety of cancer types, and is generally associated with advanced disease progression and poorer clinical outcomes, but the underlying mechanisms are unclear. Here, we show that transduction and stable expression of B7x in multiple syngeneic tumor models leads to the expansion of immunosuppressive regulatory T cells (Tregs). Mechanistically, B7x does not cause increased proliferation of Tregs in tumors, but instead promotes the conversion of conventional CD4+ T cells into Tregs. Further, we find that B7x induces global transcriptomic changes in Tregs, driving these cells to adopt an activated and suppressive phenotype. B7x increases the expression of the Treg-specific transcription factor Foxp3 in CD4+ T cells by modulating the Akt/Foxo pathway. B7x-mediated regulation of Tregs reduces the efficacy of anti-CTLA-4 treatment, a therapeutic that partially relies on Treg-depletion. However, combination treatment of anti-B7x and anti-CTLA-4 leads to synergistic therapeutic efficacy and overcomes the B7x-mediated resistance to anti-CTLA-4. Altogether, B7x mediates an immunosuppressive Treg-promoting pathway within tumors and is a promising candidate for combination immunotherapy.

PD-L1 and B7-1 Cis-Interaction: New Mechanisms in Immune Checkpoints and Immunotherapies.

Immune checkpoints negatively regulate immune cell responses. Programmed cell death protein 1:programmed death ligand 1 (PD-1:PD-L1) and cytotoxic T lymphocyte-associated protein 4 (CTLA-4):B7-1 are among the most important immune checkpoint pathways, and are key targets for immunotherapies that seek to modulate the balance between stimulatory and inhibitory signals to lead to favorable therapeutic outcomes. The current dogma of these two immune checkpoint pathways has regarded them as independent with no interactions. However, the newly characterized PD-L1:B7-1 ligand-ligand cis-interaction and its ability to bind CTLA-4 and CD28, but not PD-1, suggests that these pathways have significant crosstalk. Here, we propose that the PD-L1:B7-1 cis-interaction brings novel mechanistic understanding of these pathways, new insights into mechanisms of current immunotherapies, and fresh ideas to develop better treatments in a variety of therapeutic settings.

A Virion-Based Assay for Glycoprotein Thermostability Reveals Key Determinants of Filovirus Entry and Its Inhibition.

Ebola virus (EBOV) entry into cells is mediated by its spike glycoprotein (GP). Following attachment and internalization, virions traffic to late endosomes where GP is cleaved by host cysteine proteases. Cleaved GP then binds its cellular receptor, Niemann-Pick C1. In response to an unknown cellular trigger, GP undergoes conformational rearrangements that drive fusion of viral and endosomal membranes. The temperature-dependent stability (thermostability) of the prefusion conformers of class I viral fusion glycoproteins, including those of filovirus GPs, has provided insights into their propensity to undergo fusion-related rearrangements. However, previously described assays have relied on soluble glycoprotein ectodomains. Here, we developed a simple enzyme-linked immunosorbent assay (ELISA)-based assay that uses the temperature-dependent loss of conformational epitopes to measure thermostability of GP embedded in viral membranes. The base and glycan cap subdomains of all filovirus GPs tested suffered a concerted loss of prefusion conformation at elevated temperatures but did so at different temperature ranges, indicating virus-specific differences in thermostability. Despite these differences, all of these GPs displayed reduced thermostability upon cleavage to GP conformers (GPCL). Surprisingly, acid pH enhanced, rather than decreased, GP thermostability, suggesting it could enhance viral survival in hostile endo/lysosomal compartments. Finally, we confirmed and extended previous findings that some small-molecule inhibitors of filovirus entry destabilize EBOV GP and uncovered evidence that the most potent inhibitors act through multiple mechanisms. We establish the epitope-loss ELISA as a useful tool for studies of filovirus entry, engineering of GP variants with enhanced stability for use in vaccine development, and discovery of new stability-modulating antivirals.IMPORTANCE The development of Ebola virus countermeasures is challenged by our limited understanding of cell entry, especially at the step of membrane fusion. The surface-exposed viral protein, GP, mediates membrane fusion and undergoes major structural rearrangements during this process. The stability of GP at elevated temperatures (thermostability) can provide insights into its capacity to undergo these rearrangements. Here, we describe a new assay that uses GP-specific antibodies to measure GP thermostability under a variety of conditions relevant to viral entry. We show that proteolytic cleavage and acid pH have significant effects on GP thermostability that shed light on their respective roles in viral entry. We also show that the assay can be used to study how small-molecule entry inhibitors affect GP stability. This work provides a simple and readily accessible assay to engineer stabilized GP variants for antiviral vaccines and to discover and improve drugs that act by modulating GP stability.

Complement Protein C1q Enhances Macrophage Foam Cell Survival and Efferocytosis.

In the atherosclerotic lesion, macrophages ingest high levels of damaged modified low-density lipoproteins (LDLs), generating macrophage foam cells. Foam cells undergo apoptosis and, if not efficiently cleared by efferocytosis, can undergo secondary necrosis, leading to plaque instability and rupture. As a component of the innate immune complement cascade, C1q recognizes and opsonizes modified forms of LDL, such as oxidized or acetylated LDL, and promotes ingestion by macrophages in vitro. C1q was shown to be protective in an atherosclerosis model in vivo. Therefore, this study aimed to investigate whether ingestion of modified LDL in the presence of C1q alters macrophage foam cell survival or function. In an unbiased transcriptome analysis, C1q was shown to modulate expression of clusters of genes involved in cell death and apoptosis pathways in human monocyte-derived macrophages ingesting modified LDL; this was validated by quantitative PCR in human and murine macrophages. C1q downregulated levels and activity of active caspase-3 and PARP-1 in human and mouse macrophages during ingestion of modified LDL. This led to a measurable increase in survival and decrease in cell death, as measured by alamarBlue and propidium iodide assays, respectively. C1q opsonization also increased phagocytosis and efferocytosis in macrophage foam cells. These data suggest that C1q promotes macrophage survival during ingestion of excess cholesterol, as well as improves foam cell efferocytic function. This may be important in slowing disease progression and provides insight into the protective role of C1q in early atherosclerosis.