Moesin activation controls bone resorption and tunneling nanotube-dependent osteoclast fusion.

Osteoclasts are multinucleated cells unique in their ability to resorb bone. Osteoclastogenesis involves several steps of actin-driven rearrangements that participate not only in the cell-cell fusion process, but also in the formation of the sealing zone, the adhesive structure determining the resorption area. Despite the importance of these actin cytoskeleton-based processes, their precise mechanisms of regulation are still poorly characterized. Here, we found that moesin, a member of the Ezrin/Radixin/Moesin (ERM) protein family, is activated during osteoclast maturation and plays an instrumental role for both osteoclast fusion and function. In mouse and human osteoclast precursors, moesin is negatively regulated to potentiate their ability to fuse and degrade bone. Accordingly, we demonstrated that moesin depletion decreases membrane-to-cortex attachment and enhances formation of tunneling nanotubes (TNTs), F-actin-containing intercellular bridges that we revealed to trigger osteoclast fusion. In addition, via a ß3-integrin/RhoA/SLK pathway and independently of its role in fusion, moesin regulates the number and organization of sealing zones in mature osteoclast, and thus participates in the control of bone resorption. Supporting these findings, we found that moesin-deficient mice are osteopenic with a reduced density of trabecular bones and increased osteoclast abundance and activity. These findings provide a better understanding of the regulation of osteoclast biology, and open new opportunities to specifically target osteoclast activity in bone disease therapy.

Heteroionic Interfaces in Hybrid Solid-State Batteries─Current Constriction at the Interface between Different Solid Electrolytes.

The requirements for suitable electrolyte materials in solid-state batteries are diverse and vary greatly depending on their role as separator or as part of the composite cathode. Hybrid cell concepts that incorporate different types of solid electrolytes are considered a promising solution to overcome the limitations of single material classes. However, the kinetics at the heteroionic interface (i.e., charge transfer) substantially affects the cell performance. Moreover, non-ideal physical contacts hinder detailed electrochemical characterization of the interface properties. Thus, we use microstructure-resolved electric network computations to explore how the impedance response of a homogeneous bilayer system is influenced by the interface morphology and the material parameters of the single solid electrolyte layers. Porous interfaces and the resulting current constriction effects give rise to signatures in the impedance spectrum that resemble that of actual migration processes. This hinders unequivocal identification of the origin of the impedance contributions. The resistance and capacitance of this geometric interface signal depend strongly on the contact area and its spatial distribution, the pore capacitance, and the local conductivities around the interface. An experimental case study of an oxide-sulfide multilayer is considered to highlight the challenges in impedance analysis and the assessment of reliable material parameters. These findings are universal and apply to any heterojunction.

Rapid, concerted switching of the neural code in inferotemporal cortex.

A fundamental paradigm in neuroscience is the concept of neural coding through tuning functions 1 . According to this idea, neurons encode stimuli through fixed mappings of stimulus features to firing rates. Here, we report that the tuning of visual neurons can rapidly and coherently change across a population to attend to a whole and its parts. We set out to investigate a longstanding debate concerning whether inferotemporal (IT) cortex uses a specialized code for representing specific types of objects or whether it uses a general code that applies to any object. We found that face cells in macaque IT cortex initially adopted a general code optimized for face detection. But following a rapid, concerted population event lasting < 20 ms, the neural code transformed into a face-specific one with two striking properties: (i) response gradients to principal detection-related dimensions reversed direction, and (ii) new tuning developed to multiple higher feature space dimensions supporting fine face discrimination. These dynamics were face specific and did not occur in response to objects. Overall, these results show that, for faces, face cells shift from detection to discrimination by switching from an object-general code to a face-specific code. More broadly, our results suggest a novel mechanism for neural representation: concerted, stimulus-dependent switching of the neural code used by a cortical area.

The effect of repeated hot water immersion on insulin sensitivity, heat shock protein 70, and inflammation in individuals with type 2 diabetes mellitus.

Repeated hot water immersion (HWI) can improve glycemic control in healthy individuals but data are limited for individuals with type 2 diabetes mellitus (T2DM). The present study investigated whether repeated HWI improves insulin sensitivity and inflammatory status and reduces plasma ([extracellular heat shock protein 70]) [eHSP70] and resting metabolic rate (RMR). Fourteen individuals with T2DM participated in this pre- versus postintervention study, with outcome measures assessed in fasted (≥12 h) and postprandial (2-h post-75 g glucose ingestion) states. HWI consisted of 1 h in 40°C water (target rectal temperature 38.5°C-39°C) repeated 8-10 times within a 14-day period. Outcome measures included insulin sensitivity, plasma [glucose], [insulin], [eHSP70], inflammatory markers, RMR, and substrate utilization. The HWI intervention increased fasted insulin sensitivity (QUICKI; P = 0.03) and lowered fasted plasma [insulin] (P = 0.04), but fasting plasma [glucose] (P = 0.83), [eHSP70] (P = 0.08), [IL-6] (P = 0.55), [IL-10] (P = 0.59), postprandial insulin sensitivity (P = 0.19), plasma [glucose] (P = 0.40), and [insulin] (P = 0.47) were not different. RMR was reduced by 6.63% (P < 0.05), although carbohydrate (P = 0.43) and fat oxidation (P = 0.99) rates were unchanged. This study shows that 8-10 HWIs within a 14-day period improved fasting insulin sensitivity and plasma [insulin] in individuals with T2DM, but not when glucose tolerance is challenged. HWI also improves metabolic efficiency (i.e., reduced RMR). Together these results could be clinically important and have implications for metabolic health outcomes and well-being in individuals with T2DM.NEW & NOTEWORTHY This is the first study to investigate repeated HWI to raise deep body temperature on insulin sensitivity, inflammation, eHSP70, and substrate utilization in individuals with T2DM. The principal novel findings were improvements in fasting insulin sensitivity and fasting plasma [insulin] but no change in fasting plasma [glucose], postprandial insulin sensitivity, plasma [insulin], or [glucose]. There was also no change in eHSP70, inflammatory status, or substrate utilization but there were reductions in RMR and oxygen consumption.

Influence of Microstructure on the Material Properties of LLZO Ceramics Derived by Impedance Spectroscopy and Brick Layer Model Analysis.

Variants of garnet-type Li7La3Zr2O12 are being intensively studied as separator materials in solid-state battery research. The material-specific transport properties, such as bulk and grain boundary conductivity, are of prime interest and are mostly investigated by impedance spectroscopy. Data evaluation is usually based on the one-dimensional (1D) brick layer model, which assumes a homogeneous microstructure of identical grains. Real samples show microstructural inhomogeneities in grain size and porosity due to the complex behavior of grain growth in garnets that is very sensitive to the sintering protocol. However, the true microstructure is often omitted in impedance data analysis, hindering the interlaboratory reproducibility and comparability of results reported in the literature. Here, we use a combinatorial approach of structural analysis and three-dimensional (3D) transport modeling to explore the effects of microstructure on the derived material-specific properties of garnet-type ceramics. For this purpose, Al-doped Li7La3Zr2O12 pellets with different microstructures are fabricated and electrochemically characterized. A machine learning-assisted image segmentation approach is used for statistical analysis and quantification of the microstructural changes during sintering. A detailed analysis of transport through statistically modeled twin microstructures demonstrates that the transport parameters derived from a 1D brick layer model approach show uncertainties up to 150%, only due to variations in grain size. These uncertainties can be even larger in the presence of porosity. This study helps to better understand the role of the microstructure of polycrystalline electroceramics and its influence on experimental results.

Assessment of spilled toxic bait by wild pigs and potential risk to non-target species.

BACKGROUND: In 2018, a sodium nitrite (SN)-based toxic bait for invasive wild pigs (hereafter wild pigs; Sus scrofa), was evaluated to determine its effectiveness in reducing local wild pig populations in Texas. Localized population reductions of >70% were achieved, but spillage of bait outside wild pig-specific feeders (bait stations) caused by feeding wild pigs resulted in the deaths of non-target animals. To evaluate risks to non-target animals, we tested whether bait presentation influenced the total amount of bait spilled by wild pigs and estimated the associated risk to non-target species. RESULTS: We found that bait spilled outside bait stations could be reduced by >90% when compacted in trays, as opposed to being manually crumbled into pieces. We documented a mean spill rate of 0.913 g of bait per wild pig. Conservative risk assessments for nine non-target species for which SN toxicity data exist indicate that there is relatively low risk of lethal exposure, apart from zebra finches (Taeniopygia guttata) and white mice. Our results indicate that there may be enough spilled bait per feeding wild pig to kill 9.5 or 3.5 individuals of these species, respectively. Other species assessed range from 0.002 to 0.406 potential mortalities per wild pig. CONCLUSION: We demonstrated that the amount of bait spilled by wild pigs during feeding and the associated risk to non-target animals can be minimized by presenting the bait compacted in trays within bait stations. We recommend that baits be tightly compacted and secured in bait stations to minimize risks to non-target animals from spilled bait by wild pigs. © 2023 Society of Chemical Industry. This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA.

Understanding the Impedance of Mesoporous Oxides: Reliable Determination of the Material-Specific Conductivity.

The unique architecture of ordered mesoporous oxides makes them a promising class of materials for various electrochemical applications, such as gas sensing or energy storage and conversion. The high accessibility of the internal surface allows tailoring of their electrochemical properties, e.g., by adjusting the pore size or surface functionalization, resulting in superior device performance compared to nanoparticles or disordered mesoporous counterparts. However, optimization of the mesoporous architecture requires reliable electrochemical characterization of the system. Unfortunately, the interplay between nanocrystalline grains, grain boundaries, and the open pore framework hinders a simple estimation of material-specific transport quantities by using impedance spectroscopy. Here, we use a 3D electric network model to elucidate the impact of the pore structure on the electrical transport properties of mesoporous thin films. It is demonstrated that the impedance response is dominated only by the geometric current constriction effect arising from the regular pore network. Estimating the effective conductivity from the total resistance and the electrode geometry, thus, differs by more than 1 order of magnitude from the material-specific conductivity of the solid mesoporous framework. A detailed analysis of computed impedances for varying pore size allows for the correlation of the effective conductivity with the material-specific conductivity. We derive an empirical expression that accounts for the porous structure of the thin films and allows a reliable determination of the material-specific conductivity with an error of less than 8%.

Functional modules for visual scene segmentation in macaque visual cortex.

Segmentation, the computation of object boundaries, is one of the most important steps in intermediate visual processing. Previous studies have reported cells across visual cortex that are modulated by segmentation features, but the functional role of these cells remains unclear. First, it is unclear whether these cells encode segmentation consistently since most studies used only a limited variety of stimulus types. Second, it is unclear whether these cells are organized into specialized modules or instead randomly scattered across the visual cortex: the former would lend credence to a functional role for putative segmentation cells. Here, we used fMRI-guided electrophysiology to systematically characterize the consistency and spatial organization of segmentation-encoding cells across the visual cortex. Using fMRI, we identified a set of patches in V2, V3, V3A, V4, and V4A that were more active for stimuli containing figures compared to ground, regardless of whether figures were defined by texture, motion, luminance, or disparity. We targeted these patches for single-unit recordings and found that cells inside segmentation patches were tuned to both figure-ground and borders more consistently across types of stimuli than cells in the visual cortex outside the patches. Remarkably, we found clusters of cells inside segmentation patches that showed the same border-ownership preference across all stimulus types. Finally, using a population decoding approach, we found that segmentation could be decoded with higher accuracy from segmentation patches than from either color-selective or control regions. Overall, our results suggest that segmentation signals are preferentially encoded in spatially discrete patches.

Large-scale high-density brain-wide neural recording in nonhuman primates.

High-density, integrated silicon electrodes have begun to transform systems neuroscience, by enabling large-scale neural population recordings with single cell resolution. Existing technologies, however, have provided limited functionality in nonhuman primate species such as macaques, which offer close models of human cognition and behavior. Here, we report the design, fabrication, and performance of Neuropixels 1.0-NHP, a high channel count linear electrode array designed to enable large-scale simultaneous recording in superficial and deep structures within the macaque or other large animal brain. These devices were fabricated in two versions: 4416 electrodes along a 45 mm shank, and 2496 along a 25 mm shank. For both versions, users can programmatically select 384 channels, enabling simultaneous multi-area recording with a single probe. We demonstrate recording from over 3000 single neurons within a session, and simultaneous recordings from over 1000 neurons using multiple probes. This technology represents a significant increase in recording access and scalability relative to existing technologies, and enables new classes of experiments involving fine-grained electrophysiological characterization of brain areas, functional connectivity between cells, and simultaneous brain-wide recording at scale.

Mice and primates use distinct strategies for visual segmentation.

The rodent visual system has attracted great interest in recent years due to its experimental tractability, but the fundamental mechanisms used by the mouse to represent the visual world remain unclear. In the primate, researchers have argued from both behavioral and neural evidence that a key step in visual representation is 'figure-ground segmentation', the delineation of figures as distinct from backgrounds. To determine if mice also show behavioral and neural signatures of figure-ground segmentation, we trained mice on a figure-ground segmentation task where figures were defined by gratings and naturalistic textures moving counterphase to the background. Unlike primates, mice were severely limited in their ability to segment figure from ground using the opponent motion cue, with segmentation behavior strongly dependent on the specific carrier pattern. Remarkably, when mice were forced to localize naturalistic patterns defined by opponent motion, they adopted a strategy of brute force memorization of texture patterns. In contrast, primates, including humans, macaques, and mouse lemurs, could readily segment figures independent of carrier pattern using the opponent motion cue. Consistent with mouse behavior, neural responses to the same stimuli recorded in mouse visual areas V1, RL, and LM also did not support texture-invariant segmentation of figures using opponent motion. Modeling revealed that the texture dependence of both the mouse's behavior and neural responses could be explained by a feedforward neural network lacking explicit segmentation capabilities. These findings reveal a fundamental limitation in the ability of mice to segment visual objects compared to primates.

Psi promotes Drosophila wing growth via direct transcriptional activation of cell cycle targets and repression of growth inhibitors.

The first characterised FUSE Binding Protein family member, FUBP1, binds single-stranded DNA to activate MYC transcription. Psi, the sole FUBP protein in Drosophila, binds RNA to regulate P-element and mRNA splicing. Our previous work revealed pro-growth functions for Psi, which depend, in part, on transcriptional activation of Myc. Genome-wide functions for FUBP family proteins in transcriptional control remain obscure. Here, through the first genome-wide binding and expression profiles obtained for a FUBP family protein, we demonstrate that, in addition to being required to activate Myc to promote cell growth, Psi also directly binds and activates stg to couple growth and cell division. Thus, Psi knockdown results in reduced cell division in the wing imaginal disc. In addition to activating these pro-proliferative targets, Psi directly represses transcription of the growth inhibitor tolkin (tok, a metallopeptidase implicated in TGFß signalling). We further demonstrate tok overexpression inhibits proliferation, while tok loss of function increases mitosis alone and suppresses impaired cell division caused by Psi knockdown. Thus, Psi orchestrates growth through concurrent transcriptional activation of the pro-proliferative genes Myc and stg, in combination with repression of the growth inhibitor tok.

3D Impedance Modeling of Metal Anodes in Solid-State Batteries-Incompatibility of Pore Formation and Constriction Effect in Physical-Based 1D Circuit Models.

A non-ideal contact at the electrode/solid electrolyte interface of a solid-state battery arising due to pores (voids) or inclusions results in a geometric constriction effect that severely deteriorates the electric transport properties of the battery cell. The lack of understanding of this phenomenon hinders the optimization process of novel components, such as reversible and high-rate metal anodes. Deeper insight into the constriction phenomenon is necessary to correctly monitor interface degradation and to accelerate the successful use of metal anodes in solid-state batteries. Here, we use a 3D electric network model to study the fundamentals of the constriction effect. Our findings suggest that dynamic constriction as a non-local effect cannot be captured by conventional 1D equivalent circuit models and that its electric behavior is not ad hoc predictable. It strongly depends on the interplay of the geometry of the interface causing the constriction and the microscopic transport processes in the adjacent phases. In the presence of constriction, the contribution from the non-ideal electrode/solid electrolyte interface to the impedance spectrum may exhibit two signals that cannot be explained when the porous interface is described by a physical-based (effective medium theory) 1D equivalent circuit model. In consequence, the widespread assumption of a single interface contribution to the experimental impedance spectrum may be entirely misleading and can cause serious misinterpretation.

Interplay of Dynamic Constriction and Interface Morphology between Reversible Metal Anode and Solid Electrolyte in Solid State Batteries.

In an all-solid-state battery, the electrical contact between its individual components is of key relevance in addition to the electrochemical stability of its interfaces. Impedance spectroscopy is particularly suited for the non-destructive investigation of interfaces and of their stability under load. Establishing a valid correlation between microscopic processes and the macroscopic impedance signal, however, is challenging and prone to errors. Here, we use a 3D electric network model to systematically investigate the effect of various electrode/sample interface morphologies on the impedance spectrum. It is demonstrated that the interface impedance generally results from a charge transfer step and a geometric constriction contribution. The weights of both signals depend strongly on the material parameters as well as on the interface morphology. Dynamic constriction results from a non-ideal local contact, e.g., from pores or voids, which reduce the electrochemical active surface area only in a certain frequency range. Constriction effects dominate the interface behavior for systems with small charge transfer resistance like garnet-type solid electrolytes in contact with a lithium metal electrode. An in-depth analysis of the origin and the characteristics of the constriction phenomenon and their dependence on the interface morphology is conducted. The discussion of the constriction effect provides further insight into the processes at the microscopic level, which are, e.g., relevant in the case of reversible metal anodes.

Elexacaftor-Tezacaftor-Ivacaftor improves exercise capacity in adolescents with cystic fibrosis.

OBJECTIVE: Elexacaftor/Tezacaftor/Ivacaftor is a cystic fibrosis transmembrane conductance regulator (CFTR) modulator with the potential to improve exercise capacity. This case series of three adolescents with CF aimed to investigate whether 6 weeks treatment with Elexacaftor/Tezacaftor/Ivacaftor could improve exercise capacity in CFTR modulator naive adolescents with CF. METHODS: Three adolescents (14.0 ± 1.4 years) with CF (FEV1 % predicted: 62.5 ± 17.1; F508del/F508del genotype) completed an exhaustive maximal cardiopulmonary exercise test on a cycle ergometer to determine peak oxygen uptake ( V ̇ $\dot{{\rm{V}}}$ O2peak ) and measure changes in gas exchange and ventilation during exercise at 6 weeks. We also analyzed wrist-worn device-based physical activity (PA) data in two of the three cases. Validated acceleration thresholds were used to quantify time spent in each PA intensity category. RESULTS: Clinically meaningful improvements in V ̇ $\dot{{\rm{V}}}$ O2peak were observed in all three cases (+17.6%, +52.4%, and +32.9%, respectively), with improvements greatest in those with more severe lung disease and lower fitness at baseline. Although lung function increased in all cases, inconsistent changes in markers of ventilatory and peripheral muscle efficiency likely suggest different mechanisms of improvement in this case group of adolescents with CF. Device-based analysis of PA was variable, with one case increasing and one case decreasing. CONCLUSION: In this case series, we have observed, for the first time, improvements in exercise capacity following 6 weeks of treatment with Elexacaftor/Tezacaftor/Ivacaftor. Improvements were greatest in the presence of more severe CF lung disease and lower aerobic fitness at baseline. The mechanism(s) responsible for these changes warrant further investigation in larger trials.

Ab initiodescription of disorder effects in layered cathode active materials by the coherent potential approximation.

Disorder effects in alloys are usually modeled by averaging various supercell calculations considering different positions of the alloy atoms. This approach, however, is only possible as long as the portion of the individual components of the alloy is sufficiently large. Herein, we present anab initiostudy considering the lithium insertion material Li1-x[Ni0.33Co0.33Mn0.33]O2as model system to demonstrate the power of the coherent potential approximation within the Korringa-Kohn-Rostoker Green's function method. This approach enables the description of disorder effects within alloy systems of any composition. It is applied in this study to describe the (de-)intercalation of arbitrary amounts of lithium from the cathode active material. Moreover, we highlight that using either fully optimized structures or experimental lattice parameters and atomic positions both lead to comparable results. Our findings suggest that this approach is also suitable for modeling the electronic structure of state-of-the-art materials such as high-nickel alloys.

Stromal Notch ligands foster lymphopenia-driven functional plasticity and homeostatic proliferation of naive B cells.

In lymphopenic environments, secondary lymphoid organs regulate the size of B and T cell compartments by supporting the homeostatic proliferation of mature lymphocytes. The molecular mechanisms underlying these responses and their functional consequences remain incompletely understood. To evaluate homeostasis of the mature B cell pool during lymphopenia, we turned to an adoptive transfer model of purified follicular B cells into Rag2-/- mouse recipients. Highly purified follicular B cells transdifferentiated into marginal zone-like B cells when transferred into Rag2-/- lymphopenic hosts but not into wild-type hosts. In lymphopenic spleens, transferred B cells gradually lost their follicular phenotype and acquired characteristics of marginal zone B cells, as judged by cell surface phenotype, expression of integrins and chemokine receptors, positioning close to the marginal sinus, and an ability to rapidly generate functional plasma cells. Initiation of follicular to marginal zone B cell transdifferentiation preceded proliferation. Furthermore, the transdifferentiation process was dependent on Notch2 receptors in B cells and expression of Delta-like 1 Notch ligands by splenic Ccl19-Cre+ fibroblastic stromal cells. Gene expression analysis showed rapid induction of Notch-regulated transcripts followed by upregulated Myc expression and acquisition of broad transcriptional features of marginal zone B cells. Thus, naive mature B cells are endowed with plastic transdifferentiation potential in response to increased stromal Notch ligand availability during lymphopenia.

CFTR limits F-actin formation and promotes morphological alignment with flow in human lung microvascular endothelial cells.

Micro- and macrovascular endothelial dysfunction in response to shear stress has been observed in cystic fibrosis (CF), and has been associated with inflammation and oxidative stress. We tested the hypothesis that the cystic fibrosis transmembrane conductance regulator (CFTR) regulates endothelial actin cytoskeleton dynamics and cellular alignment in response to flow. Human lung microvascular endothelial cells (HLMVEC) were cultured with either the CFTR inhibitor GlyH-101 (20 µM) or CFTRinh-172 (20 µM), tumor necrosis factor (TNF)-α (10 ng/ml) or a vehicle control (0.1% dimethyl sulfoxide) during 24 and 48 h of exposure to shear stress (11.1 dynes/cm2 ) or under static control conditions. Cellular morphology and filamentous actin (F-actin) were assessed using immunocytochemistry. [Nitrite] and endothelin-1 ([ET-1]) were determined in cell culture supernatant by ozone-based chemiluminescence and ELISA, respectively. Treatment of HLMVECs with both CFTR inhibitors prevented alignment of HLMVEC in the direction of flow after 24 and 48 h of shear stress, compared to vehicle control (both p < 0.05). Treatment with TNF-α significantly increased total F-actin after 24 h versus control (p < 0.05), an effect that was independent of shear stress. GlyH-101 significantly increased F-actin after 24 h of shear stress versus control (p < 0.05), with a significant (p < 0.05) reduction in cortical F-actin under both static and flow conditions. Shear stress decreased [ET-1] after 24 h (p < 0.05) and increased [nitrite] after 48 h (p < 0.05), but neither [nitrite] nor [ET-1] was affected by GlyH-101 (p > 0.05). CFTR appears to limit cytosolic actin polymerization, while maintaining a cortical rim actin distribution that is important for maintaining barrier integrity and promoting alignment with flow, without effects on endothelial nitrite or ET-1 production.

Cryptosporidium rhoptry effector protein ROP1 injected during invasion targets the host cytoskeletal modulator LMO7.

The parasite Cryptosporidium invades and replicates in intestinal epithelial cells and is a leading cause of diarrheal disease and early childhood mortality. The molecular mechanisms that underlie infection and pathogenesis are largely unknown. Here, we delineate the events of host cell invasion and uncover a mechanism unique to Cryptosporidium. We developed a screen to identify parasite effectors, finding the injection of multiple parasite proteins into the host from the rhoptry organelle. These factors are targeted to diverse locations within the host cell and its interface with the parasite. One identified effector, rhoptry protein 1 (ROP1), accumulates in the terminal web of enterocytes through direct interaction with the host protein LIM domain only 7 (LMO7) an organizer of epithelial cell polarity and cell-cell adhesion. Genetic ablation of LMO7 or ROP1 in mice or parasites, respectively, impacts parasite burden in vivo in opposite ways. Taken together, these data provide molecular insight into how Cryptosporidium manipulates its intestinal host niche.

Antigen-independent activation enhances the efficacy of 4-1BB-costimulated CD22 CAR T cells.

While CD19-directed chimeric antigen receptor (CAR) T cells can induce remission in patients with B cell acute lymphoblastic leukemia (ALL), a large subset relapse with CD19- disease. Like CD19, CD22 is broadly expressed by B-lineage cells and thus serves as an alternative immunotherapy target in ALL. Here we present the composite outcomes of two pilot clinical trials ( NCT02588456 and NCT02650414 ) of T cells bearing a 4-1BB-based, CD22-targeting CAR in patients with relapsed or refractory ALL. The primary end point of these studies was to assess safety, and the secondary end point was antileukemic efficacy. We observed unexpectedly low response rates, prompting us to perform detailed interrogation of the responsible CAR biology. We found that shortening of the amino acid linker connecting the variable heavy and light chains of the CAR antigen-binding domain drove receptor homodimerization and antigen-independent signaling. In contrast to CD28-based CARs, autonomously signaling 4-1BB-based CARs demonstrated enhanced immune synapse formation, activation of pro-inflammatory genes and superior effector function. We validated this association between autonomous signaling and enhanced function in several CAR constructs and, on the basis of these observations, designed a new short-linker CD22 single-chain variable fragment for clinical evaluation. Our findings both suggest that tonic 4-1BB-based signaling is beneficial to CAR function and demonstrate the utility of bedside-to-bench-to-bedside translation in the design and implementation of CAR T cell therapies.