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The protein kinase TBK1 is a central regulator of innate immune responses and autophagy, and ablation of either function has been linked to neuroinflammatory or degenerative diseases. Autophagy is an intracellular process that recycles old or damaged proteins and organelles. In recent years, the TBK1-dependent regulation of autophagy pathways has been characterized. However, the autophagy-dependent regulation of TBK1 activity awaits further clarification. Here, we observed that TBK1 is recruited to SQSTM1/p62-containing aggregates via the selective autophagy receptor TAX1BP1. In these aggregates, TBK1 phosphorylates SQSTM1/p62 at serine 403 and thus presumably regulates the efficient engulfment and clearance of these structures. We found that TBK1 activation is strongly increased if FIP200, a component of the autophagy-inducing ULK1 complex, is not present or cannot bind to TAX1BP1. Given our collective findings, we hypothesize that FIP200 ensures the inducible activation of TBK1 at SQSTM1/p62 condensates.

Agata Smogorzewska and colleagues report mutations in SLX4 in a new subtype of Fanconi anemia. SLX4 is an endonuclease involved in DNA maintenance and repair. Fanconi anemia is a rare recessive disorder characterized by genome instability, congenital malformations, progressive bone marrow failure and predisposition to hematologic malignancies and solid tumors 1 . At the cellular level, hypersensitivity to DNA interstrand crosslinks is the defining feature in Fanconi anemia 2 . Mutations in thirteen distinct Fanconi anemia genes 3 have been shown to interfere with the DNA-replication–dependent repair of lesions involving crosslinked DNA at stalled replication forks 4 . Depletion of SLX4, which interacts with multiple nucleases and has been recently identified as a Holliday junction resolvase 5 , 6 , 7 , results in increased sensitivity of the cells to DNA crosslinking agents. Here we report the identification of biallelic SLX4 mutations in two individuals with typical clinical features of Fanconi anemia and show that the cellular defects in these individuals' cells are complemented by wildtype SLX4, demonstrating that biallelic mutations in SLX4 (renamed here as FANCP ) cause a new subtype of Fanconi anemia, Fanconi anemia-P.

Alfons Meindl and colleagues report heterozygous germline mutations in RAD51C in families with breast and ovarian cancer. Mutations were found in 1.3% of 480 pedigrees with breast and ovarian cancer, but not in 620 pedigrees with breast cancer only. Germline mutations in a number of genes involved in the recombinational repair of DNA double-strand breaks are associated with predisposition to breast and ovarian cancer. RAD51C is essential for homologous recombination repair, and a biallelic missense mutation can cause a Fanconi anemia–like phenotype. In index cases from 1,100 German families with gynecological malignancies, we identified six monoallelic pathogenic mutations in RAD51C that confer an increased risk for breast and ovarian cancer. These include two frameshift-causing insertions, two splice-site mutations and two nonfunctional missense mutations. The mutations were found exclusively within 480 pedigrees with the occurrence of both breast and ovarian tumors (BC/OC; 1.3%) and not in 620 pedigrees with breast cancer only or in 2,912 healthy German controls. These results provide the first unambiguous evidence of highly penetrant mutations associated with human cancer in a RAD51 paralog and support the 'common disease, rare allele' hypothesis.

Cellular immunotherapy using chimeric antigen receptors (CARs) so far has almost exclusively used autologous peripheral blood-derived T cells as immune effector cells. However, harvesting sufficient numbers of T cells is often challenging in heavily pre-treated patients with malignancies and perturbed hematopoiesis and perturbed hematopoiesis. Also, such a CAR product will always be specific for the individual patient. In contrast, NK cell infusions can be performed in non-HLA-matched settings due to the absence of alloreactivity of these innate immune cells. Still, the infused NK cells are subject to recognition and rejection by the patient’s immune system, thereby limiting their life-span in vivo and undermining the possibility for multiple infusions. Here, we designed genome editing and advanced lentiviral transduction protocols to render primary human NK cells unsusceptible/resistant to an allogeneic response by the recipient’s CD8 + T cells. After knocking-out surface expression of HLA class I molecules by targeting the B2M gene via CRISPR/Cas9, we also co-expressed a single-chain HLA-E molecule, thereby preventing NK cell fratricide of B2M-knockout (KO) cells via “missing self”-induced lysis. Importantly, these genetically engineered NK cells were functionally indistinguishable from their unmodified counterparts with regard to their phenotype and their natural cytotoxicity towards different AML cell lines. In co-culture assays, B2M-KO NK cells neither induced immune responses of allogeneic T cells nor re-activated allogeneic T cells which had been expanded/primed using irradiated PBMNCs of the respective NK cell donor. Our study demonstrates the feasibility of genome editing in primary allogeneic NK cells to diminish their recognition and killing by mismatched T cells and is an important prerequisite for using non-HLA-matched primary human NK cells as readily available, “off-the-shelf” immune effectors for a variety of immunotherapy indications in human cancer.

BRCA2 is an essential genome stability gene that has various functions in cells, including roles in homologous recombination, G2 checkpoint control, protection of stalled replication forks, and promotion of cellular resistance to numerous types of DNA damage. Heterozygous mutation of BRCA2 is associated with an increased risk of developing cancers of the breast, ovaries, pancreas, and other sites, thus BRCA2 acts as a classic tumor suppressor gene. However, understanding BRCA2 function as a tumor suppressor is severely limited by the fact that ~70% of the encoded protein has not been tested or assigned a function in the cellular DNA damage response. Remarkably, even the specific role(s) of many known domains in BRCA2 are not well characterized, predominantly because stable expression of the very large BRCA2 protein in cells, for experimental purposes, is challenging. Here, we review what is known about these domains and the assay systems that are available to study the cellular roles of BRCA2 domains in DNA damage responses. We also list criteria for better testing systems because, ultimately, functional assays for assessing the impact of germline and acquired mutations identified in genetic screens are important for guiding cancer prevention measures and for tailored cancer treatments.

Human mesenchymal stromal cell (MSC)-derived extracellular vesicles (EV) revealed neuroprotective potentials in various brain injury models, including neonatal encephalopathy caused by hypoxia-ischemia (HI). However, for clinical translation of an MSC-EV therapy, scaled manufacturing strategies are required, which is challenging with primary MSCs due to inter- and intra-donor heterogeneities. Therefore, we established a clonally expanded and immortalized human MSC line (ciMSC) and compared the neuroprotective potential of their EVs with EVs from primary MSCs in a murine model of HI-induced brain injury. In vivo activities of ciMSC-EVs were comprehensively characterized according to their proposed multimodal mechanisms of action. Nine-day-old C57BL/6 mice were exposed to HI followed by repetitive intranasal delivery of primary MSC-EVs or ciMSC-EVs 1, 3, and 5 days after HI. Sham-operated animals served as healthy controls. To compare neuroprotective effects of both EV preparations, total and regional brain atrophy was assessed by cresyl-violet-staining 7 days after HI. Immunohistochemistry, western blot, and real-time PCR were performed to investigate neuroinflammatory and regenerative processes. The amount of peripheral inflammatory mediators was evaluated by multiplex analyses in serum samples. Intranasal delivery of ciMSC-EVs and primary MSC-EVs comparably protected neonatal mice from HI-induced brain tissue atrophy. Mechanistically, ciMSC-EV application reduced microglia activation and astrogliosis, endothelial activation, and leukocyte infiltration. These effects were associated with a downregulation of the pro-inflammatory cytokine IL-1 beta and an elevated expression of the anti-inflammatory cytokines IL-4 and TGF-beta in the brain, while concentrations of cytokines in the peripheral blood were not affected. ciMSC-EV-mediated anti-inflammatory effects in the brain were accompanied by an increased neural progenitor and endothelial cell proliferation, oligodendrocyte maturation, and neurotrophic growth factor expression. Our data demonstrate that ciMSC-EVs conserve neuroprotective effects of primary MSC-EVs via inhibition of neuroinflammation and promotion of neuroregeneration. Since ciMSCs can overcome challenges associated with MSC heterogeneity, they appear as an ideal cell source for the scaled manufacturing of EV-based therapeutics to treat neonatal and possibly also adult brain injury.

Extracellular vesicles (EVs) can be harvested from cell culture supernatants and from all body fluids. EVs can be conceptually classified based on their size and biogenesis as exosomes and microvesicles. Nowadays, it is however commonly accepted in the field that there is a much higher degree of heterogeneity within these two subgroups than previously thought. For instance, the surface marker profile of EVs is likely dependent on the cell source, the cell's activation status, and multiple other parameters. Within recent years, several new methods and assays to study EV heterogeneity in terms of surface markers have been described; most of them are being based on flow cytometry. Unfortunately, such methods generally require dedicated instrumentation, are time-consuming and demand extensive operator expertise for sample preparation, acquisition, and data analysis. In this study, we have systematically evaluated and explored the use of a multiplex bead-based flow cytometric assay which is compatible with most standard flow cytometers and facilitates a robust semi-quantitative detection of 37 different potential EV surface markers in one sample simultaneously. First, assay variability, sample stability over time, and dynamic range were assessed together with the limitations of this assay in terms of EV input quantity required for detection of differently abundant surface markers. Next, the potential effects of EV origin, sample preparation, and quality of the EV sample on the assay were evaluated. The findings indicate that this multiplex bead-based assay is generally suitable to detect, quantify, and compare EV surface signatures in various sample types, including unprocessed cell culture supernatants, cell culture-derived EVs isolated by different methods, and biological fluids. Furthermore, the use and limitations of this assay to assess heterogeneities in EV surface signatures was explored by combining different sets of detection antibodies in EV samples derived from different cell lines and subsets of rare cells. Taken together, this validated multiplex bead-based flow cytometric assay allows robust, sensitive, and reproducible detection of EV surface marker expression in various sample types in a semi-quantitative way and will be highly valuable for many researchers in the EV field in different experimental contexts.

Christopher Mathew and colleagues report a homozygous germline mutation of RAD51C in a Fanconi anemia-like disorder. Mutation of RAD51C , encoding a protein involved in homologous recombination-mediated DNA repair, leads to hypersensitivity to DNA cross-linking agents. Fanconi anemia (FA) is a rare chromosomal-instability disorder associated with a variety of developmental abnormalities, bone marrow failure and predisposition to leukemia and other cancers 1 . We have identified a homozygous missense mutation in the RAD51C gene in a consanguineous family with multiple severe congenital abnormalities characteristic of FA. RAD51C is a member of the RAD51-like gene family involved in homologous recombination–mediated DNA repair. The mutation results in loss of RAD51 focus formation in response to DNA damage and in increased cellular sensitivity to the DNA interstrand cross-linking agent mitomycin C and the topoisomerase-1 inhibitor camptothecin. Thus, biallelic germline mutations in a RAD51 paralog are associated with an FA-like syndrome.

The great clinical success of chimeric antigen receptor (CAR) T cells has unlocked new levels of immunotherapy for hematological malignancies. Genetically modifying natural killer (NK) cells as alternative CAR immune effector cells is also highly promising, as NK cells can be transplanted across HLA barriers without causing graft-versus-host disease. Therefore, off-the-shelf usage of CAR NK cell products might allow to widely expand the clinical indications and to limit the costs of treatment per patient. However, in contrast to T cells, manufacturing suitable CAR NK cell products is challenging, as standard techniques for genetically engineering NK cells are still being defined. In this study, we have established optimal lentiviral transduction of primary human NK cells by systematically testing different internal promoters for lentiviral CAR vectors and comparing lentiviral pseudotypes and viral entry enhancers. We have additionally modified CAR constructs recognizing standard target antigens for acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML) therapy—CD19, CD33, and CD123—to harbor a CD34-derived hinge region that allows efficient detection of transduced NK cells in vitro and in vivo and also facilitates CD34 microbead-assisted selection of CAR NK cell products to >95% purity for potential clinical usage. Importantly, as most leukemic blasts are a priori immunogenic for activated primary human NK cells, we developed an in vitro system that blocks the activating receptors NKG2D, DNAM-1, NKp30, NKp44, NKp46, and NKp80 on these cells and therefore allows systematic testing of the specific killing of CAR NK cells against ALL and AML cell lines and primary AML blasts. Finally, we evaluated in an ALL xenotransplantation model in NOD/SCID-gamma (NSG) mice whether human CD19 CAR NK cells directed against the CD19+ blasts are relying on soluble or membrane-bound IL15 production for NK cell persistence and also in vivo leukemia control. Hence, our study provides important insights into the generation of pure and highly active allogeneic CAR NK cells, thereby advancing adoptive cellular immunotherapy with CAR NK cells for human malignancies further.

Despite advancements in cancer therapies, bacterial complications remain a major challenge, delaying treatment and worsening outcomes. While immunosuppressive therapies and prolonged hospitalizations contribute, they do not fully explain the elevated infection risk in cancer patients. Here we show that tumors producing high levels of granulocyte colony-stimulating factor (G-CSF) promote the persistence of Gram-negative pathogens in head and neck squamous cell carcinoma due to neutrophil reprogramming. Mechanistically, we identify tumor-driven activation of the G-CSF / nicotinamide phosphoribosyltransferase (NAMPT) signaling axis in neutrophil progenitors, resulting in impaired antibacterial functions, such as phagocytosis and neutrophil extracellular traps formation, and development of tissue-damaging neutrophil subsets. This disrupts lung tissue integrity and facilitates bacterial persistence. Importantly, targeting the G-CSF/NAMPT pathway prevents the generation of dysfunctional neutrophils and improves bacterial clearance in vivo. Our findings reveal tumor-induced, NAMPT-dependent neutrophil reprogramming as a central driver of compromised antimicrobial defenses in cancer. Therapeutic strategies aimed at modulating G-CSF/NAMPT signaling could enhance infection control and survival for cancer patients. Cancer patients are at increased risk for severe bacterial infections due to immune dysfunction. Here, the authors show that chronic tumor-derived G-CSF drives NAMPT/NAD-dependent neutrophil dysfunction from the progenitor stage, and that targeting this pathway restores infection control.