Chimeric antigen receptor (CAR)–T cells have shown efficacy in patients with B cell malignancies. Yet, their application for solid tumors has challenges that include limited cancer-specific targets and nonpersistence of adoptively transferred CAR-T cells. Here, we introduce the developmentally regulated tight junction protein claudin 6 (CLDN6) as a CAR target in solid tumors and a strategy to overcome inefficient CAR-T cell stimulation in vivo. We demonstrate that a nanoparticulate RNA vaccine, designed for body-wide delivery of the CAR antigen into lymphoid compartments, stimulates adoptively transferred CAR-T cells. Presentation of the natively folded target on resident antigen-presenting cells promotes cognate and selective expansion of CAR-T cells. Improved engraftment of CAR-T cells and regression of large tumors in difficult-to-treat mouse models was achieved at subtherapeutic CAR-T cell doses.

Plasticity of the resilient keratin intermediate filament cytoskeleton is an important prerequisite for epithelial tissue homeostasis. Here, the contribution of stress-activated p38 MAPK to keratin network organization was examined in cultured cells. It was observed that phosphorylated p38 colocalized with keratin granules that were rapidly formed in response to orthovanadate. The same p38p recruitment was noted during mitosis, in various stress situations and in cells producing mutant keratins. In all these situations keratin 8 became phosphorylated on S73, a well-known p38 target site. To demonstrate that p38-dependent keratin phosphorylation determines keratin organization, p38 activity was pharmacologically and genetically modulated: up-regulation induced keratin granule formation, whereas down-regulation prevented keratin filament network disassembly. Furthermore, transient p38 inhibition also inhibited keratin filament precursor formation and mutant keratin granule dissolution. Collectively, the rapid and reversible effects of p38 activity on keratin phosphorylation and organization in diverse physiological, stress, and pathological situations identify p38-dependent signalling as a major intermediate filament–regulating pathway.

Recent studies showed that keratin filament (KF) formation originates primarily from sites close to the actin-rich cell cortex. To further characterize these sites, we performed multicolor fluorescence imaging of living cells and found drastically increased KF assembly in regions of elevated actin turnover, i.e., in lamellipodia. Abundant KF precursors (KFPs) appeared within these areas at the distal tips of actin stress fibers, moving alongside the stress fibers until their integration into the peripheral KF network. The earliest KFPs were detected next to actin-anchoring focal adhesions (FAs) and were only seen after the establishment of FAs in emerging lamellipodia. Tight spatiotemporal coupling of FAs and KFP formation were not restricted to epithelial cells, but also occurred in nonepithelial cells and cells producing mutant keratins. Finally, interference with FA formation by talin short hairpin RNA led to KFP depletion. Collectively, our results support a major regulatory function of FAs for KF assembly, thereby providing the basis for coordinated shaping of the entire cytoskeleton during cell relocation and rearrangement.

It is generally assumed that turnover of the keratin filament system occurs by exchange of subunits along its entire length throughout the cytoplasm. We now present evidence that a circumscribed submembranous compartment is actually the main site for network replenishment. This conclusion is based on the following observations in living cells synthesizing fluorescent keratin polypeptides: 1) Small keratin granules originate in close proximity to the plasma membrane and move toward the cell center in a continuous motion while elongating into flexible rod-like fragments that fuse with each other and integrate into the peripheral KF network. 2) Recurrence of fluorescence after photobleaching is first seen in the cell periphery where keratin filaments are born that translocate subsequently as part of the network toward the cell center. 3) Partial keratin network reformation after orthovanadate-induced disruption is restricted to a distinct peripheral zone in which either keratin granules or keratin filaments are transiently formed. These findings extend earlier investigations of mitotic cells in which de novo keratin network formation was shown to originate from the cell cortex. Taken together, our results demonstrate that the keratin filament system is not homogeneous but is organized into temporally and spatially distinct subdomains. Furthermore, the cortical localization of the regulatory cues for keratin filament turnover provides an ideal way to adjust the epithelial cytoskeleton to dynamic cellular processes.

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