SIV and HIV-based envelope V1-deleted (ΔV1) vaccines, delivered systemically by the DNA/ALVAC/gp120 platform, decrease the risk of mucosal SIV or SHIV acquisition more effectively than V1-replete vaccines. Here we investigated the induction of mucosal and systemic memory-like NK cells as well as antigen-reactive ILC response by DNA/ALVAC/gp120-based vaccination and their role against SIV/SHIV infection. ΔV1 HIV vaccination elicited a higher level of mucosal TNF-α+ and CD107+ memory-like NK cells than V1-replete vaccination, suggesting immunogen dependence. Mucosal memory-like NK cells, systemic granzyme B+ memory NK cells, and vaccine-induced mucosal envelope antigen-reactive IL-17+ NKp44+ ILCs, IL-17+ ILC3s, and IL-13+ ILC2 subsets were linked to a lower risk of virus acquisition. Additionally, mucosal memory-like NK cells and mucosal env-reactive IFN-γ+ ILC1s and env- reactive IL-13+ ILC2 subsets correlated with viral load control. We further observed a positive correlation between post-vaccination systemic and mucosal memory-like NK cells, suggesting vaccination enhances the presence of these cells in both compartments. Mucosal and systemic memory-like NK cells positively correlated with V2-specific ADCC responses, a reproducible correlate of reduced risk of SIV/HIV infection. In contrast, an increased risk was associated with the level of mucosal PMA/Ionomycin-induced IFN-γ+ and CD107+ NKG2A-NKp44- ILCs. Plasma proteomic analyses demonstrated that suppression of mucosal memory-like NK cells was linked to the level of CCL-19, LT-α, TNFSF-12, and IL-15, suppression of systemic env-reactive granzyme B+ memory-like NK cells was associated with the level of OLR1, CCL-3, and OSM, and suppression of IL-17+ ILCs immunity was correlated with the level of IL-6 and CXCL-9. In contrast, FLT3 ligand was associated with promotion of protective mucosal env-reactive IL-17+ responses. These findings emphasize the importance of mucosal memory-like NK cell and envelope- reactive ILC responses for protection against mucosal SIV/SHIV acquisition.
Copyright © 2024 Rahman, Silva de Castro, Schifanella, Bissa and Franchini.

Head and neck squamous cell carcinoma (HNSCC) is a global cancer burden with a 5-year overall survival rate of around 50%, stagnant for decades. A tumour-induced immunosuppressive microenvironment contributes to HNSCC progression, with the adenosine (ADO) pathway and an upregulated expression of inhibitory immune checkpoint regulators playing a key role in this context. The correlation between high neutrophil-to-lymphocyte ratio (NLR) with advanced tumour staging suggests involvement of neutrophils (NØ) in cancer progression. Interestingly, we associated a high NLR with an increased intracellular PD-L1 localization in primary HNSCC samples, potentially mediating more aggressive tumour characteristics and therefore synergistically favouring tumour progression. Still, further research is needed to harness this knowledge for effective treatments and overcome resistance. Since it is hypothesized that the tumour microenvironment (TME) may be influenced by small extracellular vesicles (sEVs) secreted by tumours (TEX), this study aims to investigate the impact of HNSCC-derived TEX on NØ and blockade of ADO receptors as a potential strategy to reverse the pro-tumour phenotype of NØ. UMSCC47-TEX exhibited CD73 enzymatic activity involved in ADO signalling, as well as the immune checkpoint inhibitor PD-L1. Data revealed that TEX induce chemotaxis of NØ and the sustained interaction promotes a shift into a pro-tumour phenotype, dependent on ADO receptors (P1R), increasing CD170high subpopulation, CD73 and PD-L1 expression, followed by an immunosuppressive secretome. Blocking A3R reduced CD73 and PD-L1 expression. Co-culture experiments with HNSCC cells demonstrated that TEX-modulated NØ increase the CD73/PD-L1 axis, through Cyclin D-CDK4/6 signalling. To support these findings, the CAM model with primary tumour was treated with NØ supernatant. Moreover, these NØ promoted an increase in migration, invasion, and reduced cell death. Targeting P1R on NØ, particularly A3R, exhibited potential therapeutic strategy to counteract immunosuppression in HNSCC. Understanding the TEX-mediated crosstalk between tumours and NØ offers insights into immunomodulation for improving cancer therapies.
© 2024 The Authors. Journal of Extracellular Vesicles published by Wiley Periodicals, LLC on behalf of the International Society for Extracellular Vesicles.

Septic patients with worst clinical prognosis have increased circulating immature granulocytes (IG), displaying limited phagocytosis and reactive oxygen species (ROS) production. Here, we developed an ex vivo model of incubation of human granulocytes, from septic patients or healthy donors, with Escherichia coli. We showed that the ROS production in Sepsis-IG is lower due to decreased activation and protein expression of the NADPH oxidase complex. We also demonstrated that the low level of ROS production and lower phagocytosis of IG in sepsis induce the bacterial SOS response, leading to the expression of the SOS-regulated quinolone resistance gene qnrB2. Without antimicrobial pressure, the sepsis immune response alone may promote antibiotic resistance expression.
© 2024 The Authors.

Severe congenital neutropenia (CN) is an inherited pre-leukemia bone marrow failure syndrome commonly caused by autosomal-dominant ELANE mutations (ELANE-CN). ELANE-CN patients are treated with daily injections of recombinant human granulocyte colony-stimulating factor (rhG-CSF). However, some patients do not respond to rhG-CSF, and approximately 15% of ELANE-CN patients develop myelodysplasia or acute myeloid leukemia. Here, we report the development of a curative therapy for ELANE-CN through inhibition of ELANE mRNA expression by introducing two single-strand DNA breaks at the opposing DNA strands of the ELANE promoter TATA box using CRISPR-Cas9D10A nickases-termed MILESTONE. This editing effectively restored defective neutrophil differentiation of ELANE-CN CD34+ hematopoietic stem and progenitor cells (HSPCs) in vitro and in vivo, without affecting the functions of the edited neutrophils. CRISPResso analysis of the edited ELANE-CN CD34+ HSPCs revealed on-target efficiencies of over 90%. Simultaneously, GUIDE-seq, CAST-Seq, and rhAmpSeq indicated a safe off-target profile with no off-target sites or chromosomal translocations. Taken together, ex vivo gene editing of ELANE-CN HSPCs using MILESTONE in the setting of autologous stem cell transplantation could be a universal, safe, and efficient gene therapy approach for ELANE-CN patients.
Copyright © 2024 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved.

The appearance of senescent cells in age-related diseases has spurred the search for compounds that can target senescent cells in tissues, termed senolytics. However, a major caveat with current senolytic screens is the use of cell lines as targets where senescence is induced in vitro, which does not necessarily reflect the identity and function of pathogenic senescent cells in vivo. Here, we developed a new pipeline leveraging a fluorescent murine reporter that allows for isolation and quantification of p16Ink4a+ cells in diseased tissues. By high-throughput screening in vitro, precision-cut lung slice (PCLS) screening ex vivo, and phenotypic screening in vivo, we identified a HSP90 inhibitor, XL888, as a potent senolytic in tissue fibrosis. XL888 treatment eliminated pathogenic p16Ink4a+ fibroblasts in a murine model of lung fibrosis and reduced fibrotic burden. Finally, XL888 preferentially targeted p16INK4a-hi human lung fibroblasts isolated from patients with idiopathic pulmonary fibrosis (IPF), and reduced p16INK4a+ fibroblasts from IPF PCLS ex vivo. This study provides proof of concept for a platform where p16INK4a+ cells are directly isolated from diseased tissues to identify compounds with in vivo and ex vivo efficacy in mice and humans, respectively, and provides a senolytic screening platform for other age-related diseases.