Human B Cells Mediate Innate Anti-Cancer Cytotoxicity Through Concurrent Engagement of Multiple TNF Superfamily Ligands.

The essential innate immunity effector cells, natural killer and dendritic cells, express multiple plasma membrane-associated tumor necrosis factor (TNF) superfamily (TNFSF) ligands that, through simultaneous and synergistic engagement, mediate anti-cancer cytotoxicity. Here, we report that circulating B cells, mediators of adaptive humoral immunity, also mediate this innate anti-cancer immune mechanism. We show that resting human B cells isolated from peripheral blood induce apoptosis of, and efficiently kill a large variety of leukemia and solid tumor cell types. Single-cell RNA sequencing analyses indicate, and flow cytometry data confirm that B cells from circulation express transmembrane TNF, Fas ligand (FasL), lymphotoxin (LT) α1ß2 and TNF-related apoptosis-inducing ligand (TRAIL). The cytotoxic activity can be inhibited by individual and, especially, combined blockade of the four transmembrane TNFSF ligands. B cells from tumor-bearing head and neck squamous cell carcinoma patients express lower levels of TNFSF ligands and are less cytotoxic than those isolated from healthy individuals. In conclusion, we demonstrate that B cells have the innate capacity to mediate anti-cancer cytotoxicity through concurrent activity of multiple plasma membrane-associated TNFSF ligands, that this mechanism is deficient in cancer patients and that it may be part of a general cancer immunosurveillance mechanism.

Sex Differences Revealed in a Mouse CFA Inflammation Model with Macrophage Targeted Nanotheranostics.

Monocyte derived macrophages (MDMs) infiltrate sites of infection or injury and upregulate cyclooxygenase-2 (COX-2), an enzyme that stimulates prostaglandin-E2 (PgE2). Nanotheranostics combine therapeutic and diagnostic agents into a single nanosystem. In previous studies, we demonstrated that a nanotheranostic strategy, based on theranostic nanoemulsions (NE) loaded with a COX-2 inhibitor (celecoxib, CXB) and equipped with near-infrared fluorescent (NIRF) reporters, can specifically target circulating monocytes and MDMs. The anti-inflammatory and anti-nociceptive effects of such cell-specific COX-2 inhibition lasted several days following Complete Freund's Adjuvant (CFA) or nerve injury in male mice. The overall goal of this study was to investigate the extended (up to 40 days) impact of MDM-targeted COX-2 inhibition and any sex-based differences in treatment response; both of which remain unknown. Our study also evaluates the feasibility and efficacy of a preclinical nanotheranostic strategy for mechanistic investigation of the impact of such sex differences on clinical outcomes. Methods: CFA was administered into the right hind paws of male and female mice. All mice received a single intravenous dose of NIRF labeled CXB loaded NE twelve hours prior to CFA injection. In vivo whole body NIRF imaging and mechanical hypersensitivity assays were performed sequentially and ex vivo NIRF imaging and immunohistopathology of foot pad tissues were performed at the end point of 40 days. Results: Targeted COX-2 inhibition of MDMs in male and female mice successfully improved mechanical hypersensitivity after CFA injury. However, we observed distinct sex-specific differences in the intensity or longevity of the nociceptive responses. In males, a single dose of CXB-NE administered via tail vein injection produced significant improved mechanical hypersensitivity for 32 days as compared to the drug free NE (DF-NE) (untreated) control group. In females, CXB-NE produced similar, though less prominent and shorter-lived effects, lasting up to 11 days. NIRF imaging confirmed that CXB-NE can be detected up to day 40 in the CFA injected foot pad tissues of both sexes. There were distinct signal distribution trends between males and females, suggesting differences in macrophage infiltration dynamics between the sexes. This may also relate to differences in macrophage turnover rate between the sexes, a possibility that requires further investigation in this model. Conclusions: For the first time, this study provides unique insight into MDM dynamics and the early as well as longer-term targeted effects and efficacy of a clinically translatable nanotheranostic agent on MDM mediated inflammation. Our data supports the potential of nanotheranostics as presented in elucidating the kinetics, dynamics and sex-based differences in the adaptive or innate immune responses to inflammatory triggers. Taken together, our study findings lead us closer to true personalized, sex-specific pain nanomedicine for a wide range of inflammatory diseases.

An altered redox balance mediates the hypersensitivity of Cockayne syndrome primary fibroblasts to oxidative stress.

Cockayne syndrome (CS) is a rare hereditary multisystem disease characterized by neurological and development impairment, and premature aging. Cockayne syndrome cells are hypersensitive to oxidative stress, but the molecular mechanisms involved remain unresolved. Here we provide the first evidence that primary fibroblasts derived from patients with CS-A and CS-B present an altered redox balance with increased steady-state levels of intracellular reactive oxygen species (ROS) and basal and induced DNA oxidative damage, loss of the mitochondrial membrane potential, and a significant decrease in the rate of basal oxidative phosphorylation. The Na/K-ATPase, a relevant target of oxidative stress, is also affected with reduced transcription in CS fibroblasts and normal protein levels restored upon complementation with wild-type genes. High-resolution magnetic resonance spectroscopy revealed a significantly perturbed metabolic profile in CS-A and CS-B primary fibroblasts compared with normal cells in agreement with increased oxidative stress and alterations in cell bioenergetics. The affected processes include oxidative metabolism, glycolysis, choline phospholipid metabolism, and osmoregulation. The alterations in intracellular ROS content, oxidative DNA damage, and metabolic profile were partially rescued by the addition of an antioxidant in the culture medium suggesting that the continuous oxidative stress that characterizes CS cells plays a causative role in the underlying pathophysiology. The changes of oxidative and energy metabolism offer a clue for the clinical features of patients with CS and provide novel tools valuable for both diagnosis and therapy.

Innate direct anticancer effector function of human immature dendritic cells. I. Involvement of an apoptosis-inducing pathway.

Dendritic cells (DCs) mediate cross-priming of tumor-specific T cells by acquiring tumor Ags from dead cancer cells. The process of cross-priming would be most economical and efficient if DCs also induce death of cancer cells. In this study, we demonstrate that normal human in vitro generated immature DCs consistently and efficiently induce apoptosis in cancer cell lines, freshly isolated noncultured cancer cells, and normal proliferating endothelial cells, but not in most normal cells. In addition, in vivo generated noncultured peripheral blood immature DCs mediate similar tumoricidal activity as their in vitro counterpart, indicating that this DC activity might be biologically relevant. In contrast to immature DCs, freshly isolated monocytes (myeloid DC precursors) and in vitro generated mature DCs are not cytotoxic or are less cytotoxic, respectively, suggesting that DC-mediated killing of cancer cells is developmentally regulated. Comparable cytotoxic activity is mediated by untreated DCs, paraformaldehyde-fixed DCs, and soluble products of DCs, and is destructible by proteases, indicating that both cell membrane-bound and secreted proteins mediate this DC function. Overall, our data demonstrate that human immature DCs are capable of inducing apoptosis in cancer cells and thus to both directly mediate anticancer activity and initiate processing of cellular tumor Ags.

Innate direct anticancer effector function of human immature dendritic cells. II. Role of TNF, lymphotoxin-alpha(1)beta(2), Fas ligand, and TNF-related apoptosis-inducing ligand.

Our recent studies have demonstrated that human immature dendritic cells (DCs) are able to directly and effectively mediate apoptotic killing against a wide array of cultured and freshly-isolated cancer cells without harming normal cells. In the present study, we demonstrate that this tumoricidal activity is mediated by multiple cytotoxic TNF family ligands. We determine that human immature DCs express on their cell surface four different cytotoxic TNF family ligands: TNF, lymphotoxin-alpha(1)beta(2), Fas ligand, and TNF-related apoptosis inducing ligand; while cancer cells express the corresponding death receptors. Disruptions of interactions between the four ligands expressed on DCs and corresponding death-signaling receptors expressed on cancer cells using specific Abs or R:Fc fusion proteins block the cytotoxic activity of DCs directed against cancer cells. The novel findings suggest that DC killing of cancer cells is mediated by the concerted engagement of four TNF family ligands of DCs with corresponding death receptors of cancer cells. Overall, our data demonstrate that DCs are fully equipped for an efficient direct apoptotic killing of cancer cells and suggest that this mechanism may play a critical role in both afferent and efferent anti-tumor immunity.