TUMOR-INTRINSIC INFLAMMATORY PATHWAYS ASSOCIATED WITH TUMOR DORMANCY AND RECURRENCE

The successful treatment of breast cancer is limited due to a fraction of tumor cells escaping drug-treatment by entering a dormant state, only to relapse years or decades later at distant sites. Host-driven chronic inflammatory cells such as M2 macrophages play an important role in tumorigenesis, but the role of tumor-intrinsic inflammatory signaling involved in tumor dormancy and recurrence is unknown. We sought to determine the role of tumor-intrinsic inflammatory pathways in mouse mammary carcinoma cells (MMC) treated with Adriamycin (ADR), a clinically relevant chemotherapeutic drug. We found that ADR-induced dormant tumor cells autonomously produced pro-inflammatory cytokines, in vitro. MMC treated with Chloroquine (CQ) prior to ADR treatment displayed a delay in relapse, or prolonging of dormancy, when compared to ADR-treated MMC. Additional gene array data showed predicated activation of NF-κB p65 in ADR-treated dormant MMC that eventually relapsed. These data suggest that the anti-inflammatory function of CQ led to prolonged dormancy. To test this, we investigated the role of inflammatory signaling pathways directly by shRNA-mediated knockdown and CRISPR-Cas9-mediated knockout of NF-κB p65 in MMC. We found that knockdown of NF-κB p65 resulted in fewer dormant cells after ADR treatment and reduced rate of relapse, in vitro. NF-κB p65 was also found to reduce the immunomodulatory effects of ADR, with shNF-κB p65 showing increased upregulation of neu upon ADR treatment. Additionally, we found NF-κB p65 to be associated with a higher infiltration of CD8+ T cells and anti-tumor T cell responses. Our findings suggest a dual role of tumor-intrinsic NF-κB p65 pathway, allowing for escape from drug treatment through dormancy which leads to relapse, but also for proper lymphocyte infiltration and subsequent anti-tumor activity

Toward high-throughput engineering techniques for improving CAR intracellular signaling domains

Chimeric antigen receptors (CAR) are generated by linking extracellular antigen recognition domains with one or more intracellular signaling domains derived from the T-cell receptor complex or various co-stimulatory receptors. The choice and relative positioning of signaling domains help to determine chimeric antigen receptors T-cell activity and fate in vivo. While prior studies have focused on optimizing signaling power through combinatorial investigation of native intracellular signaling domains in modular fashion, few have investigated the prospect of sequence engineering within domains. Here, we sought to develop a novel in situ screening method that could permit deployment of directed evolution approaches to identify intracellular domain variants that drive selective induction of transcription factors. To accomplish this goal, we evaluated a screening approach based on the activation of a human NF-κB and NFAT reporter T-cell line for the isolation of mutations that directly impact T cell activation in vitro. As a proof-of-concept, a model library of chimeric antigen receptors signaling domain variants was constructed and used to demonstrate the ability to discern amongst chimeric antigen receptors containing different co-stimulatory domains. A rare, higher-signaling variant with frequency as low as 1 in 1000 could be identified in a high throughput setting. Collectively, this work highlights both prospects and limitations of novel mammalian display methods for chimeric antigen receptors signaling domain discovery and points to potential strategies for future chimeric antigen receptors development

Antibody attributes that predict the neutralization and effector function of polyclonal responses to SARS-CoV-2

BACKGROUND: While antibodies can provide significant protection from SARS-CoV-2 infection and disease sequelae, the specific attributes of the humoral response that contribute to immunity are incompletely defined. METHODS: We employ machine learning to relate characteristics of the polyclonal antibody response raised by natural infection to diverse antibody effector functions and neutralization potency with the goal of generating both accurate predictions of each activity based on antibody response profiles as well as insights into antibody mechanisms of action. RESULTS: To this end, antibody-mediated phagocytosis, cytotoxicity, complement deposition, and neutralization were accurately predicted from biophysical antibody profiles in both discovery and validation cohorts. These models identified SARS-CoV-2-specific IgM as a key predictor of neutralization activity whose mechanistic relevance was supported experimentally by depletion. CONCLUSIONS: Validated models of how different aspects of the humoral response relate to antiviral antibody activities suggest desirable attributes to recapitulate by vaccination or other antibody-based interventions