Single-cell epigenomic dysregulation of Systemic Sclerosis fibroblasts via CREB1/EGR1 axis in self-assembled human skin equivalents.

Systemic sclerosis (SSc) is an autoimmune disease characterized by skin fibrosis, internal organ involvement and vascular dropout. We previously developed and phenotypically characterized an in vitro 3D skin-like tissue model of SSc, and now analyze the transcriptomic (scRNA-seq) and epigenetic (scATAC-seq) characteristics of this model at single-cell resolution. SSc 3D skin-like tissues were fabricated using autologous fibroblasts, macrophages, and plasma from SSc patients or healthy control (HC) donors. SSc tissues displayed increased dermal thickness and contractility, as well as increased α-SMA staining. Single-cell transcriptomic and epigenomic analyses identified keratinocytes, macrophages, and five populations of fibroblasts (labeled FB1 - 5). Notably, FB1 APOE-expressing fibroblasts were 12-fold enriched in SSc tissues and were characterized by high EGR1 motif accessibility. Pseudotime analysis suggests that FB1 fibroblasts differentiate from a TGF-ß1-responsive fibroblast population and ligand-receptor analysis indicates that the FB1 fibroblasts are active in macrophage crosstalk via soluble ligands including FGF2 and APP. These findings provide characterization of the 3D skin-like model at single cell resolution and establish that it recapitulates subsets of fibroblasts and macrophage phenotypes observed in skin biopsies.

CDDO-Methyl Ester Inhibits BRAF Inhibitor Resistance and Remodels the Myeloid Compartment in BRAF-mutant Melanoma.

Approximately 50% of advanced melanomas harbor activating BRAF V600E mutations that are sensitive to BRAF inhibition. However, the duration of the response to BRAF inhibitors (BRAFi) has been limited due to the development of acquired resistance, which is preceded by recruitment of immunosuppressive myeloid cells and regulatory T cells (T regs ). While the addition of MAPK/ERK kinase 1 inhibitors (MEKi) prolongs therapeutic response to BRAF inhibition, most patients still develop resistance. Using a Braf V600E/+ /Pten -/- graft mouse model of melanoma, we now show that the addition of the methyl ester of the synthetic triterpenoid 2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oic acid (C-Me) to the BRAFi vemurafenib analog PLX4720 at resistance significantly reduces tumor burden. Dual treatment remodels the BRAFi resistant-tumor microenvironment (TME), reducing infiltration of T regs and tumor associated macrophages (TAMs), and attenuates immunosuppressive cytokine production. For the first time, we characterize myeloid populations using scRNA-seq in BRAFi-resistant tumors and demonstrate that restoration of therapeutic response is associated with significant changes in immune-activated myeloid subset representation. Collectively, these studies suggest that C-Me inhibits acquired resistance to BRAFi. Use of C-Me in combination with other therapies may both inhibit melanoma growth and enhance therapeutic responsiveness more broadly.

T Cells and CDDO-Me Attenuate Immunosuppressive Activation of Human Melanoma-Conditioned Macrophages.

Melanoma tumors are highly immunogenic, making them an attractive target for immunotherapy. However, many patients do not mount robust clinical responses to targeted therapies, which is attributable, at least in part, to suppression of immune responses by tumor-associated macrophages (TAMs) in the tumor microenvironment (TME). Using a human in vitro tri-culture system of macrophages with activated autologous T cells and BRAFV600E mutant melanoma cells, we now show that activated T cells and the synthetic triterpenoid the methyl ester of 2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oic acid (CDDO-Me) attenuate immune suppression. Surface expression of CD206, CD16 and CD163 on melanoma-conditioned macrophages was inhibited by the addition of T cells, suggesting relief of immuno-suppressive macrophage activation. We also demonstrated that addition of CDDO-Me to tri-cultures enhanced T cell-mediated reductions in CCL2, VEGF and IL-6 production in a contact-independent manner. Because these results suggest CDDO-Me alters melanoma-conditioned macrophage activation, we interrogated CDDO-Me-mediated changes in macrophage signaling pathway activation. Our results indicated that CDDO-Me inhibited phosphorylation of STAT3, a known inducer of TAM activation. Collectively, our studies suggest that activated T cells and CDDO-Me synergistically relieve immune suppression in melanoma cultures and implicate the potential utility of CDDO-Me in the treatment of melanoma.

Structural Basis for Potent Neutralization of Betacoronaviruses by Single-Domain Camelid Antibodies.

Coronaviruses make use of a large envelope protein called spike (S) to engage host cell receptors and catalyze membrane fusion. Because of the vital role that these S proteins play, they represent a vulnerable target for the development of therapeutics. Here, we describe the isolation of single-domain antibodies (VHHs) from a llama immunized with prefusion-stabilized coronavirus spikes. These VHHs neutralize MERS-CoV or SARS-CoV-1 S pseudotyped viruses, respectively. Crystal structures of these VHHs bound to their respective viral targets reveal two distinct epitopes, but both VHHs interfere with receptor binding. We also show cross-reactivity between the SARS-CoV-1 S-directed VHH and SARS-CoV-2 S and demonstrate that this cross-reactive VHH neutralizes SARS-CoV-2 S pseudotyped viruses as a bivalent human IgG Fc-fusion. These data provide a molecular basis for the neutralization of pathogenic betacoronaviruses by VHHs and suggest that these molecules may serve as useful therapeutics during coronavirus outbreaks.

CDDO-Me Alters the Tumor Microenvironment in Estrogen Receptor Negative Breast Cancer.

The tumor microenvironment (TME) is an essential contributor to the development and progression of malignancy. Within the TME, tumor associated macrophages (TAMs) mediate angiogenesis, metastasis, and immunosuppression, which inhibits infiltration of tumor-specific cytotoxic CD8+ T cells. In previous work, we demonstrated that the synthetic triterpenoid CDDO-methyl ester (CDDO-Me) converts breast TAMs from a tumor-promoting to a tumor-inhibiting activation state in vitro. We show now that CDDO-Me remodels the breast TME, redirecting TAM activation and T cell tumor infiltration in vivo. We demonstrate that CDDO-Me significantly attenuates IL-10 and VEGF expression but stimulates TNF production, and reduces surface expression of CD206 and CD115, markers of immunosuppressive TAMs. CDDO-Me treatment redirects the TAM transcriptional profile, inducing signaling pathways associated with immune stimulation, and inhibits TAM tumor infiltration, consistent with decreased expression of CCL2. In CDDO-Me-treated mice, both the absolute number and proportion of splenic CD4+ T cells were reduced, while the proportion of CD8+ T cells was significantly increased in both tumors and spleen. Moreover, mice fed CDDO-Me demonstrated significant reductions in numbers of CD4+ Foxp3+ regulatory T cells within tumors. These results demonstrate for the first time that CDDO-Me relieves immunosuppression in the breast TME and unleashes host adaptive anti-tumor immunity.

Comparison of Immune Responses to Different Versions of VLP Associated Stabilized RSV Pre-Fusion F Protein.

Efforts to develop a vaccine for respiratory syncytial virus (RSV) have primarily focused on the RSV fusion protein. The pre-fusion conformation of this protein induces the most potent neutralizing antibodies and is the focus of recent efforts in vaccine development. Following the first identification of mutations in the RSV F protein (DS-Cav1 mutant protein) that stabilized the pre-fusion conformation, other mutant stabilized pre-fusion F proteins have been described. To determine if there are differences in alternate versions of stabilized pre-fusion F proteins, we explored the use, as vaccine candidates, of virus-like particles (VLPs) containing five different pre-fusion F proteins, including the DS-Cav1 protein. The expression of these five pre-F proteins, their assembly into VLPs, their pre-fusion conformation stability in VLPs, their reactivity with anti-F monoclonal antibodies, and their induction of immune responses after the immunization of mice, were characterized, comparing VLPs containing the DS-Cav1 pre-F protein with VLPs containing four alternative pre-fusion F proteins. The concentrations of anti-F IgG induced by each VLP that blocked the binding of prototype monoclonal antibodies using two different soluble pre-fusion F proteins as targets were measured. Our results indicate that both the conformation and immunogenicity of alternative VLP associated stabilized pre-fusion RSV F proteins are different from those of DS-Cav1 VLPs.

Oncogenic BRAFV600E Governs Regulatory T-cell Recruitment during Melanoma Tumorigenesis.

Regulatory T cells (Treg) are critical mediators of immunosuppression in established tumors, although little is known about their role in restraining immunosurveillance during tumorigenesis. Here, we employ an inducible autochthonous model of melanoma to investigate the earliest Treg and CD8 effector T-cell responses during oncogene-driven tumorigenesis. Induction of oncogenic BRAFV600E and loss of Pten in melanocytes led to localized accumulation of FoxP3+ Tregs, but not CD8 T cells, within 1 week of detectable increases in melanocyte differentiation antigen expression. Melanoma tumorigenesis elicited early expansion of shared tumor/self-antigen-specific, thymically derived Tregs in draining lymph nodes, and induced their subsequent recruitment to sites of tumorigenesis in the skin. Lymph node egress of tumor-activated Tregs was required for their C-C chemokine receptor 4 (Ccr4)-dependent homing to nascent tumor sites. Notably, BRAFV600E signaling controlled expression of Ccr4-cognate chemokines and governed recruitment of Tregs to tumor-induced skin sites. BRAFV600E expression alone in melanocytes resulted in nevus formation and associated Treg recruitment, indicating that BRAFV600E signaling is sufficient to recruit Tregs. Treg depletion liberated immunosurveillance, evidenced by CD8 T-cell responses against the tumor/self-antigen gp100, which was concurrent with the formation of microscopic neoplasia. These studies establish a novel role for BRAFV600E as a tumor cell-intrinsic mediator of immune evasion and underscore the critical early role of Treg-mediated suppression during autochthonous tumorigenesis.Significance: This work provides new insights into the mechanisms by which oncogenic pathways impact immune regulation in the nascent tumor microenvironment. Cancer Res; 78(17); 5038-49. ©2018 AACR.