Benita Wolf

Small-molecule probes are transformative for cell biology, offering unprecedented insights into subcellular structures, including in systems without molecular genetic tools. Centrioles are fundamental for generating the axoneme of cilia and flagella, as well as centrosomes, but a generic small-molecule probe allowing selective visualization of centriolar and axonemal microtubules is lacking. We engineered CenSpark as a cell-permeable dual-ligand fluorescent probe exploiting the juxtaposition of inner and outer microtubule-binding sites of microtubule triplets and doublets present exclusively in centriolar and axonemal microtubules. This design endows CenSpark high selectivity in live and fixed specimen analysis of centrioles, cilia and flagella across systems. We deployed CenSpark to uncover the rate of primary cilium formation and track centrioles in chimeric antigen receptor T cells during polarization at the immunological synapse with unprecedented resolution. Overall, CenSpark is a novel versatile small-molecule fluorescent probe to monitor centrioles, cilia and flagella without the need for genetic manipulation.

Given the critical role of CD4 T cells in anti-tumor immunity, strategies to harness these cells for cancer immunotherapy are gaining increasing interest. Historically overshadowed by CD8 T cells, cytotoxic CD4 T cells can directly kill MHC class II-expressing tumor cells. However, the defining molecular signature and the mechanisms underlying their cytolytic activity remain poorly understood, particularly in cancer patients. Here, using ex vivo single-cell transcriptomic and spatial analyses of CD4 T cells from paired blood and tumor samples of melanoma patients, we identified Killer Cell Lectin-Like Receptor G1 (KLRG1) as a defining surface marker of cytotoxic CD4 T cells. The CD4 ⁺ KLRG1 ⁺ T cell subset was notably enriched among circulating cells compared with tumor-infiltrating populations, which were instead enriched in T follicular helper (Tfh) states. Functionally, KLRG1 ⁺ CD4 T cells expressed elevated levels of cytotoxic genes and exhibited superior tumor-killing capacity compared with their KLRG1 ^- counterparts. We demonstrated that their cytotoxicity is granulysin-dependent, as confirmed by CRISPR/Cas9-mediated gene deletion. Mechanistically, CD4 T cells spared MHC class II ⁺ cells lacking the KLRG1 ligands CD324 and CD325, such as professional antigen-presenting cells (APCs), indicating that cytotoxicity was selectively directed towards tumor cells while preserving immune cells. Finally, by investigating how the tumor microenvironment may impair CD4 T cell cytotoxicity, we showed that tumor-derived factors, including interleukin-6 (IL-6), are key drivers promoting the transition of cytotoxic CD4 T cells toward a Tfh phenotype. In summary, our findings define KLRG1 as a defining cell surface marker of cytotoxic CD4 T cells in cancer patients, as well as a key regulator that protects MHC class II ⁺ APCs. Moreover, targeting the IL-6 signalling pathway may enhance CD4 T cell anti-tumor cytotoxicity, offering new avenues for cancer immunotherapy.

Summary: Cellular communication is critical for anti-cancer immunity, with tumor cell killing occurring at immunological synapses (IS) formed between effector immune cells and target tumor cells. While optical super-resolution microscopy (SRM) has enlightened the spatial organization of the IS mostly in regular immune cells, visualizing the nanoscale architectural features of IS in its native state, including 3D receptor distribution and the ultrastructural details of the lytic granule release remains challenging. Using cryo-expansion microscopy (cryo-ExM), we unravel the cellular architecture of activated T cells and T cell-target cell pairs. Our approach visualizes actin and microtubule networks during synapse formation, membrane topography, and the distribution of signaling molecules and lytic granules of different types, offering novel insights into IS organization. Finally, we apply U-ExM to glioblastoma tissue, visualizing T cells and their lytic content in situ , highlighting its potential for pre-clinical immunotherapy studies.

TRIM37 is an E3 ubiquitin ligase mutated in Mulibrey nanism, a disease with impaired organ growth and increased tumor formation. TRIM37 depletion from tissue culture cells results in supernumerary foci bearing the centriolar protein Centrin. Here, we characterize these centriolar protein assemblies (Cenpas) to uncover the mechanism of action of TRIM37. We find that an atypical de novo assembly pathway can generate Cenpas that act as microtubule-organizing centers (MTOCs), including in Mulibrey patient cells. Correlative light electron microscopy reveals that Cenpas are centriole-related or electron-dense structures with stripes. TRIM37 regulates the stability and solubility of Centrobin, which accumulates in elongated entities resembling the striped electron dense structures upon TRIM37 depletion. Furthermore, Cenpas formation upon TRIM37 depletion requires PLK4, as well as two parallel pathways relying respectively on Centrobin and PLK1. Overall, our work uncovers how TRIM37 prevents Cenpas formation, which would otherwise threaten genome integrity.

TRIM37 is an E3 ubiquitin ligase mutated in Mulibrey nanism, a disease characterized by impaired growth and increased tumorigenesis, whose cellular etiology is poorly understood. TRIM37 depletion from tissue culture cells results in supernumerary foci bearing the centriolar protein Centrin. Here, we characterized these centriolar protein assemblies (Cenpas) to uncover the mechanism of action of TRIM37. We established that an atypical de novo assembly pathway is notably involved in forming Cenpas, which can nevertheless trigger further centriole assembly and act as MTOCs. We found also that Cenpas are present and act similarly in Mulibrey patient cells. Through correlative light electron microscopy, we uncovered that Cenpas correspond to centriole related structures and elongated electron-dense structures with stripes. Importantly, we established that TRIM37 regulates the stability and solubility of the centriolar protein Centrobin. Our findings suggest that elongated Centrobin assemblies are a major constituent of the striped electron dense structures. Furthermore, we established that Cenpas formation upon TRIM37 depletion requires PLK4 activity, as well as two parallel pathways relying respectively on Centrobin and PLK1. Overall, our work uncovers how TRIM37 prevents the formation of Cenpas that would otherwise threaten genome integrity, including possibly in Mulibrey patients.

Background Adoptive cell therapy (ACT) using tumor-infiltrating lymphocytes (TILs) is a promising experimental immunotherapy that has shown high objective responses in patients with melanoma. Current protocols use a lymphodepletive chemotherapy before infusion of ex vivo expanded TILs, followed by high-dose interleukin-2 (IL-2). Treatment-related toxicities are mainly attributable to the chemotherapy regimen and to the high-dose IL-2 and are generally reversible. Neurological side effects have rarely been described. Nevertheless, due to improvements in cell production techniques and due to combinations with other immunomodulating molecules, side effects not previously described may be encountered. Case presentation We report the case of a 53-year-old heavily pretreated patient with melanoma who developed Guillain-Barré syndrome (GBS) 19 days after ACT using autologous TILs, given in the context of a phase I trial. He presented with dorsal back pain, unsteady gait and numbness in hands and feet. Lumbar puncture showed albuminocytological dissociation, and nerve conduction studies revealed prolonged distal motor latencies in median, ulnar, tibial and peroneal nerves, compatible with a GBS. The patient was treated with intravenous immunoglobulins and intensive neurological rehabilitation, with progressive and full recovery at 21 months post-TIL-ACT. Concomitant to the onset of GBS, a cytomegalovirus reactivation on immunosuppression was detected and considered as the most plausible cause of this neurological side effect. Conclusion We describe for the first time a case of GBS occurring shortly after TIL-ACT for melanoma, even though we could not identify with certainty the triggering agent. The report of such rare cases is of extreme importance to build on the knowledge of immune cellular therapies and their specific spectrum of toxicities.

Proper spindle positioning is crucial for spatial cell division control. Spindle positioning in human cells relies on a ternary complex comprising Gαi1–3, LGN and NuMA, which anchors dynein at the cell cortex, thus enabling pulling forces to be exerted on astral microtubules. We develop a live imaging siRNA-based screen using stereotyped fibronectin micropatterns to uncover components modulating spindle positioning in human cells, testing 1280 genes, including all kinases and phosphatases. We thus discover 16 components whose inactivation dramatically perturbs spindle positioning, including tyrosine receptor kinase 3 (TYRO3) and cyclin G associated kinase (GAK). TYRO3 depletion results in excess NuMA and dynein at the cortex during metaphase, similar to the effect of blocking the TYRO3 downstream target phosphatidylinositol 3-kinase (PI3K). Furthermore, depletion of GAK leads to impaired astral microtubules, similar to the effect of downregulating the GAK-interactor Clathrin. Overall, our work uncovers components and mechanisms governing spindle positioning in human cells.

Adoptive T cell therapy (ACT) is a safe and effective personalized cancer immunotherapy that can comprise naturally occurring ex vivo expanded cells (e.g., tumor-infiltrating lymphocytes [TIL]) or T cells genetically engineered to confer antigen specificity (T-cell receptor [TCR] or chimeric antigen receptor [CAR] engineered T cells) to mediate cancer rejection. In recent years, some ACTs have produced unprecedented breakthrough responses: TIL therapy has moved from melanoma to solid tumor applications, TCR-engineered cells are developed for hematologic and solid tumors, and CAR-engineered T cells have received Food and Drug Administration (FDA) approval for the treatment of patients with certain B-cell malignancies. Although results are encouraging, to date, only a small percentage of patients with advanced malignancies can benefit from ACT. Besides ACT availability and accessibility, treatment-related toxicities represent a major hurdle in the widespread implementation of this therapeutic modality. The large variety of observed toxicities is caused by the infused cell product or as side effects of accompanying medication and chemotherapy. Toxicities can occur immediately or can be delayed. In order to render those highly promising therapeutic approaches safe enough for a wider pool of patients outside of clinical trials, an international consensus for toxicity management needs to be established.