Altering Bacille Calmette-Guérin (BCG) immunization from low-dose intradermal (i.d.) to high-dose intravenous (i.v.) vaccination provides a high level of protection against Mycobacterium tuberculosis (Mtb). In addition to strong T cell immunity, i.v. BCG drives robust humoral immune responses that track with bacterial control. However, given the near-complete protection afforded by high-dose i.v. BCG immunization, a precise correlate of protection was difficult to define. Here we leveraged plasma and bronchoalveolar lavage fluid (BAL) from a cohort of rhesus macaques that received decreasing doses of i.v. BCG and aimed to define correlates of immunity following Mtb challenge. We show an i.v. BCG dose-dependent induction of mycobacterial-specific humoral immune responses. Antibody responses at peak immunogenicity predicted bacterial control post-challenge. Multivariate analyses revealed antibody-mediated complement and natural killer (NK) cell-activating humoral networks as key signatures of protective immunity. This work extends our understanding of humoral biomarkers and potential mechanisms of i.v. BCG-mediated protection against Mtb.
© 2024 The Authors.

Arterial endothelial cells (AECs) are the founder cells for intraembryonic haematopoiesis. Here, we report a method for the efficient generation of human haemogenic DLL4+ AECs from pluripotent stem cells (PSC). Time-series single-cell RNA-sequencing reveals the dynamic evolution of haematopoiesis and lymphopoiesis, generating cell types with counterparts present in early human embryos, including stages marked by the pre-haematopoietic stem cell genes MECOM/EVI1, MLLT3 and SPINK2. DLL4+ AECs robustly support lymphoid differentiation, without the requirement for exogenous NOTCH ligands. Using this system, we find IL7 acts as a morphogenic factor determining the fate choice between the T and innate lymphoid lineages and also plays a role in regulating the relative expression level of RAG1. Moreover, we document a developmental pathway by which human RAG1+ lymphoid precursors give rise to the natural killer cell lineage. Our study describes an efficient method for producing lymphoid progenitors, providing insights into their endothelial and haematopoietic ontogeny, and establishing a platform to investigate the development of the human blood system.
© 2024. The Author(s).

Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), remains a global health burden. While M. tuberculosis is primarily a respiratory pathogen, it can spread to other organs, including the brain and meninges, causing TB meningitis (TBM). However, little is known about the immunological mechanisms that lead to differential disease across organs. Attention has focused on differences in T cell responses in the control of M. tuberculosis in the lungs, but emerging data point to a role for antibodies, as both biomarkers of disease control and as antimicrobial molecules. Given an increasing appreciation for compartmentalized antibody responses across the blood-brain barrier, here we characterized the antibody profiles across the blood and brain compartments in TBM and determined whether M. tuberculosis-specific humoral immune responses differed between M. tuberculosis infection of the lung (pulmonary TB) and TBM. Using a high throughput systems serology approach, we deeply profiled the antibody responses against 10 different M. tuberculosis antigens, including lipoarabinomannan (LAM) and purified protein derivative (PPD), in HIV-negative adults with pulmonary TB (n = 10) versus TBM (n = 60). Antibody studies included analysis of immunoglobulin isotypes (IgG, IgM, IgA) and subclass levels (IgG1-4) and the capacity of M. tuberculosis-specific antibodies to bind to Fc receptors or C1q and to activate innate immune effector functions (complement and natural killer cell activation; monocyte or neutrophil phagocytosis). Machine learning methods were applied to characterize serum and CSF responses in TBM, identify prognostic factors associated with disease severity, and define the key antibody features that distinguish TBM from pulmonary TB. In individuals with TBM, we identified CSF-specific antibody profiles that marked a unique and compartmentalized humoral response against M. tuberculosis, characterized by an enrichment of M. tuberculosis-specific antibodies able to robustly activate complement and drive phagocytosis by monocytes and neutrophils, all of which were associated with milder TBM severity at presentation. Moreover, individuals with TBM exhibited M. tuberculosis-specific antibodies in the serum with an increased capacity to activate phagocytosis by monocytes, compared with individuals with pulmonary TB, despite having lower IgG titres and Fcγ receptor-binding capacity. Collectively, these data point to functionally divergent humoral responses depending on the site of infection (i.e. lungs versus brain) and demonstrate a highly compartmentalized M. tuberculosis-specific antibody response within the CSF in TBM. Moreover, our results suggest that phagocytosis- and complement-mediating antibodies may promote attenuated neuropathology and milder TBM disease.
© The Author(s) 2024. Published by Oxford University Press on behalf of the Guarantors of Brain.

Overexpression and aberrant activation of signal transducer and activator of transcription 3 (STAT3) contribute to tumorigenesis, drug resistance, and tumor-immune evasion, making it a potential cancer therapeutic target. BP1003 is a neutral liposome incorporated with a nuclease-resistant P-ethoxy antisense oligodeoxynucleotide (ASO) targeting the STAT3 mRNA. Its unique design enhances BP1003 stability, cellular uptake, and target affinity. BP1003 efficiently reduces STAT3 expression and enhances the sensitivity of breast cancer cells (HER2+, triple negative) and ovarian cancer cells (late stage, invasive ovarian cancer) to paclitaxel and 5-fluorouracil (5-FU) in both 2D and 3D cell cultures. Similarly, ex vivo and in vivo patient-derived models of pancreatic ductal adenocarcinoma (PDAC) show reduced tissue viability and tumor volume with BP1003 and gemcitabine combination treatments. In addition to directly affecting tumor cells, BP1003 can modulate the tumor microenvironment. Unlike M1 differentiation, monocyte differentiation into anti-inflammatory M2 macrophages is suppressed by BP1003, indicating its potential contribution to immunotherapy. The broad anti-tumor effect of BP1003 in numerous preclinical solid tumor models, such as breast, ovarian, and pancreatic cancer models shown in this work, makes it a promising cancer therapeutic.

Preclinical studies imply that surgery triggers inflammation that may entail tumor outgrowth and metastasis. The potential impact of surgery-induced inflammation in human pancreatic cancer is insufficiently explored. This study included 17 patients with periampullary cancer [pancreatic ductal adenocarcinoma (PDAC) n = 14, ampullary carcinoma n = 2, cholangiocarcinoma n = 1] undergoing major pancreatic cancer surgery with curative intent. We analyzed the potential impact of preoperative and postoperative immune phenotypes and function on postoperative survival with >30 months follow-up. The surgery entailed prompt expansion of monocytic myeloid-derived suppressor cells (M-MDSC) that generated NOX2-derived reactive oxygen species (ROS). Strong induction of immunosuppressive M-MDSC after surgery predicted poor postoperative survival and coincided with reduced functionality of circulating natural killer (NK) cells. The negative impact of surgery-induced M-MDSC on survival remained significant in separate analysis of patients with PDAC. M-MDSC-like cells isolated from patients after surgery significantly suppressed NK cell function ex vivo, which was reversed by inhibition of NOX2-derived ROS. High NOX2 subunit expression within resected tumors from patients with PDAC correlated with poor survival whereas high expression of markers of cytotoxic cells associated with longer survival. The surgery-induced myeloid inflammation was recapitulated in vivo in a murine model of NK cell-dependent metastasis. Surgical stress thus induced systemic accumulation of M-MDSC-like cells and promoted metastasis of NK cell-sensitive tumor cells. Genetic or pharmacologic suppression of NOX2 reduced surgery-induced inflammation and distant metastasis in this model. We propose that NOX2-derived ROS generated by surgery-induced M-MDSC may be targeted for improved outcome after pancreatic cancer surgery.
Pancreatic cancer surgery triggered pronounced accumulation of NOX2+ myeloid-derived suppressor cells that inhibited NK cell function and negatively prognosticated postoperative patient survival. We propose the targeting of M-MDSC as a conceivable strategy to reduce postoperative immunosuppression in pancreatic cancer.
© 2024 The Authors; Published by the American Association for Cancer Research.