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Non-thermal plasma (NTP) shows promise as a potent anti-cancer therapy with both cytotoxic and immunomodulatory effects. In this study, we investigate the chemical and biological effects of NTP-induced oxidation on several key, determinant immune checkpoints of natural killer (NK) cell function. We used molecular dynamics (MD) and umbrella sampling simulations to investigate the effect of NTP-induced oxidative changes on the MHC-I complexes HLA-Cw4 and HLA-E. Our simulations indicate that these chemical alterations do not significantly affect the binding affinity of these markers to their corresponding NK cell receptor, which is supported with experimental read-outs of ligand expression on human head and neck squamous cell carcinoma cells after NTP application. Broadening our scope to other key ligands for NK cell reactivity, we demonstrate rapid reduction in CD155 and CD112, target ligands of the inhibitory TIGIT axis, and in immune checkpoint CD73 immediately after treatment. Besides these transient chemical alterations, the reactive species in NTP cause a cascade of downstream cellular reactions. This is underlined by the upregulation of the stress proteins MICA/B, potent ligands for NK cell activation, 24 h post treatment. Taken together, this work corroborates the immunomodulatory potential of NTP, and sheds light on the interaction mechanisms between NTP and cancer cells.

Summary: Here, we present a protocol to perform CRISPR-Cas9 genome editing in human resting primary natural killer (NK) and NK-92 cells. We describe steps for guide RNA selection, ribonucleoprotein (RNP) complex formation, delivery via Nucleofection, and analysis of CRISPR edits to assess editing efficiencies. This protocol offers a tool for functional studies in NK cells, paving the way for potential applications in immunotherapy and beyond. We also discuss limitations such as off-target effects and cell-type-specific considerations. : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Summary: The role of T cell receptor (TCR) diversity in infectious disease susceptibility is not well understood. We use a systems immunology approach on three cohorts of herpes zoster (HZ) patients and controls to investigate whether TCR diversity against varicella-zoster virus (VZV) influences the risk of HZ. We show that CD4+ T cell TCR diversity against VZV glycoprotein E (gE) and immediate early 63 protein (IE63) after 1-week culture is more restricted in HZ patients. Single-cell RNA and TCR sequencing of VZV-specific T cells shows that T cell activation pathways are significantly decreased after stimulation with VZV peptides in convalescent HZ patients. TCR clustering indicates that TCRs from HZ patients co-cluster more often together than TCRs from controls. Collectively, our results suggest that not only lower VZV-specific TCR diversity but also reduced functional TCR affinity for VZV-specific proteins in HZ patients leads to lower T cell activation and consequently affects the susceptibility for viral reactivation.

Summary: Wilms’ tumor protein 1 (WT1) is a tumor-associated antigen overexpressed in various cancers. As a self-antigen, negative selection reduces the number of WT1-specific T cell receptors (TCRs). Here, we provide a protocol to generate WT137-45-specific TCRs using healthy human peripheral blood mononuclear cells. We describe the expansion of WT1-specific T cell clones by two consecutive in vitro stimulations with autologous WT137-45-pulsed dendritic cells and peripheral blood lymphocytes. We then detail the detection with human leukocyte antigen/WT137-45 tetramers. : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Gap junctions (GJs), essential structures for cell-cell communication, are made of two hemichannels (commonly called connexons), one on each adjacent cell. Found in almost all cells, GJs play a pivotal role in many physiological and cellular processes, and have even been linked to the progression of diseases, such as cancer. Modulation of GJs is under investigation as a therapeutic strategy to kill tumor cells. Furthermore, GJs have also been studied for their key role in activating anti-cancer immunity and propagating radiation- and oxidative stress-induced cell death to neighboring cells, a process known as the bystander effect. While, gap junction (GJ)-based therapeutic strategies are being developed, one major challenge has been the paradoxical role of GJs in both tumor progression and suppression, based on GJ composition, cancer factors, and tumoral context. Therefore, understanding the mechanisms of action, regulation, and the dual characteristics of GJs in cancer is critical for developing effective therapeutics. In this review, we provide an overview of the current understanding of GJs structure, function, and paradoxical pro- and anti-tumoral role in cancer. We also discuss the treatment strategies to target these GJs properties for anti-cancer responses, via modulation of GJ function.

Multiple cancer therapies nowadays rely on oxidative stress to damage cancer cells. Here we investigated the biological and molecular effect of oxidative stress on the interaction between CD44 and hyaluronan (HA), as interrupting their binding can hinder cancer progression. Our experiments demonstrated that the oxidation of HA decreased its recognition by CD44, which was further enhanced when both CD44 and HA were oxidized. The reduction of CD44–HA binding negatively affected the proliferative state of cancer cells. Our multi-level atomistic simulations revealed that the binding free energy of HA to CD44 decreased upon oxidation. The effect of HA and CD44 oxidation on CD44–HA binding was similar, but when both HA and CD44 were oxidized, the effect was much larger, in agreement with our experiments. Hence, our experiments and computations support our hypothesis on the role of oxidation in the disturbance of CD44–HA interaction, which can lead to the inhibition of proliferative signaling pathways inside the tumor cell to induce cell death.