Biology subjects

Smith, A. S.

Publications and source records attributed to Smith, A. S..

16 recordsLinked to original sources

bioRxiv2026

Genetic control couples TGFβ-independent pluripotent stem cell renewal to deterministic forward programming across germ layers

Reproducible and scalable manufacture of human induced pluripotent stem cell (hiPSC) derivatives requires control over expansion and differentiation. Transcription factor-mediated forward programming can place hiPSC differentiation under genetic control, but expansion still relies on extracellular growth-factor supplementation, increasing process complexity and operator dependence. Here we introduce GERALT (Genetically Enhanced Renewal and Autonomous Lineage Transdifferentiation), a push-pull genetic circuit assembled through targeted safe-harbour integration that couples NANOG-supported, TGF{beta}-independent renewal to doxycycline-inducible lineage programming and simultaneous NANOG silencing. GERALT maintained pluripotency during extended TGF{beta} withdrawal in both adherent and suspension culture, yet remained programmable towards myogenic, neuronal, and hepatic fates. Compared with the established OPTi-OX platform in myogenesis, GERALT enabled differentiation in simplified medium while canalising and accelerating commitment. GERALT also supported direct myogenic programming in suspension and enabled direct casting of hiPSCs into hydrogel followed by in-tissue programming, yielding electrically responsive engineered muscle tissues. These results extend genetic control upstream into stem cell renewal, facilitating deployment of new approach methodologies and other biomanufacturing applications.

synthetic biology↗

bioRxiv2025

Heterogeneity in environmental stiffness alone can guide cells and shape tissues

While topographical and chemical cues are well known to regulate cell shape and function, the role of stiffness heterogeneity has remained unclear. Here, we demonstrate - for the first time to our knowledge - that cells can be guided solely by the stiffness heterogeneity of their environment. To that end, we engineered a cell-guiding platform with abrupt, subcellular stiff and soft domains, whose flatness and uniform chemistry eliminated confounding cues. Cells elongate and align along stiff regions, sensing soft domains as barriers when wider than 2 microns. Perturbated myosin activity, cortical tension, and elasticity contrast reveal distinct biomechanical contributions, while a probabilistic model integrating adhesion, contractility, and cortical tension extracts key mechanical parameters characterizing the cellular state. Finally, experiments and dissipative particle dynamics demonstrate collective stiffness-based contact guidance. This work identifies stiffness heterogeneity as a fundamental regulator of cell and tissue organization and provides a framework for designing mechanoregulatory biomaterials.

biophysics↗

bioRxiv2025

Neoantigens drive adoptively transferred CD8 T cells to long-lived effectors mediated by lymph node trafficking

Adoptive transfer of neoantigen-reactive T lymphocytes mediates potent responses against solid tumors in patients while often limiting toxicity to normal tissues; however, the mechanisms governing the trafficking, activation, and superior antitumor function of infused T cells remain unknown. Using a clinically relevant TCR-transgenic T cell therapy model, we examined CD8 T cell responses to melanoma expressing either wild-type or mutated antigen. Neoantigen expression conferred robust tumor regression, durable cures, and long-term protective immunity. Mechanistically, T cells reacting to neoantigen exhibited enhanced cytokine and chemokine production, heightened effector function, and sustained persistence within the blood, tumor, and lymph nodes. Notably, trafficking through secondary lymphoid organs was necessary for T cell persistence and antitumor efficacy. These findings highlight the critical role of T cell trafficking to the lymph nodes in shaping neoantigen-specific antitumor responses and offers insight for improving adoptive cellular therapies.

cancer biology↗

bioRxiv2024

Opposite-sex pairing alters social interaction-induced GCaMP and dopamine activity in the insular cortex of male prairie voles

The prairie vole (Microtus ochrogaster) is a monogamous rodent species which displays selective social behaviors to conspecifics after establishing a pair bonded relationship, specifically partner-directed affiliation and stranger-directed aggression. This social selectivity relies on the ability of an individual to respond appropriately to a social context and requires salience detection and valence assignment. The anterior insular cortex (aIC) has been implicated in stimulus processing and categorization across a variety of contexts and is well-situated to integrate environmental stimuli and internal affective states to modulate complex goal-directed behaviors and social decision-making. Surprisingly, the contribution of the aIC to the expression of pair bond-induced social selectivity in prairie voles has been drastically understudied. Here we examined whether neural activity and gene expression in the aIC change in response to opposite-sex pairing and/or as a function of pairing length in male prairie voles. Opposite-sex pairing was characterized by changes to calcium and dopamine (DA) transients in the aIC that corresponded with the display of social selectivity across pair bond maturation. Furthermore, D1 and D2 receptor mRNA expression was significantly higher in males after 48 hrs of cohabitation with a female partner compared to same-sex housed males, and D2 mRNA remained significantly higher in males with a female partner compared to same-sex housed males after a week of cohabitation. Together, these results implicate a role for DA and its receptors in the aIC across the transition from early-to late-phase pair bonding.

neuroscience↗

bioRxiv2024

Extended amygdala corticotropin-releasing hormone neurons regulate sexually dimorphic changes in pair bond formation following social defeat in prairie voles (Microtus ochrogaster)

The neurobiological mechanisms underlying the connection between anxiety brought on by social stressors and the negative impact on relationship formation have remained elusive. In order to address this question, we used the social defeat model in the socially monogamous prairie vole to investigate the impact of this stress on pair bond formation. Social defeat experience inhibited partner preference formation in males but promoted preference in females. Furthermore, pair bonding increased corticotropin-releasing hormone (CRH) expression in the bed nucleus of the stria terminalis (BNST) in male prairie voles, while defeat experience increased BNST CRH expression in females. Chemogenetic excitation of BNST CRH neurons during a short cohabitation with a new partner promoted a partner preference in stress-naive prairie voles. Interestingly, chemogenetic inhibition of BNST CRH neurons during cohabitation with a new partner blocked partner preference in stress-naive males but promoted preference in defeated males. Inhibition of BNST CRH neurons also blocked partner preference in stress-naive females but did not alter preference behavior in defeated females. This study revealed sexual dimorphism in not only the impact of social defeat on pair bond formation, but also in the role BNST CRHergic neurons play in regulating changes in pair bonding following social conflict.

neuroscience↗

bioRxiv2023

The degree of T cell stemness differentially impacts the potency of adoptive cancer immunotherapy in a Lef-1 and Tcf-1 dependent manner.

Generating stem memory T cells (TSCM) is a key goal for improving cancer immunotherapy. Yet, the optimal way to modulate signaling pathways that enrich TSCM properties remains elusive. Here, we discovered that the degree to which the PI3K{delta} pathway is blocked pharmaceutically can generate T cells with differential levels of stemness properties. This observation was based on the progressive enrichment of transcriptional factors of stemness (Tcf-1 and Lef-1). Additional investigation revealed that T cells with high stemness features had enhanced metabolic plasticity, marked by heightened mitochondrial function and glucose uptake. Conversely, T cells with low or medium features of stemness expressed more inhibitory checkpoint receptors (Tim-3, CD39) and were vulnerable to antigen-induced cell death. Only TCR-antigen specific T cells with high stemness persisted following adoptive transfer in vivo and mounted protective immunity to melanoma tumors. Likewise, the strongest level of PI3K{delta} blockade in vitro generated human tumor infiltrating lymphocytes (TILs) and CAR T cells with heightened stemness properties, in turn bolstering their capacity to regress human mesothelioma tumors. We find that the level of stemness T cells possess in vitro differentially impacts their potency upon transfer in three tumor models. Mechanistically, both Lef-1 and Tcf-1 sustain anti-tumor protection by high TSCM, as deletion of either one compromised cellular therapy. Collectively, these findings highlight the therapeutic potential of carefully modulating PI3K{delta} signaling in T cells to confer high stemness and mediate protective responses to solid tumors.

immunology↗

bioRxiv2023

Functional connectivity favors hyperactivity leading to synapse loss in amyloidosis

While Alzheimers disease (AD) has been extensively studied with a focus on cognitive networks, sensory network dysfunction has received comparatively less attention despite compelling evidence of its significance in both Alzheimers disease patients and mouse models. We recently found that neurons in the primary visual cortex of an AD mouse model expressing human amyloid protein precursor with the Swedish and Indiana mutations (hAPP mutations) exhibit aberrant c-Fos expression and altered synaptic structures at a pre-amyloid plaque stage. However, it is unclear whether aberrant c-Fos expression and synaptic pathology vary across the broader visual network and to what extent c-Fos abnormality in the cortex is inherited through functional connectivity. Using both sexes of 4-6-month AD model mice with hAPP mutations (J20[PDGF-APPSw, Ind]), we found that cortical regions of the visual network show aberrant c-Fos expression and impaired experience-dependent modulation while subcortical regions do not. Interestingly, the average network-wide functional connectivity strength of a brain region in wild type (WT) mice significantly predicts its aberrant c-Fos expression, which in turn correlates with impaired experience-dependent modulation in the AD model. Using in vivo two-photon and ex vivo imaging of presynaptic termini, we observed a subtle yet selective weakening of excitatory cortical synapses in the visual cortex. Intriguingly, the change in the size distribution of cortical boutons in the AD model is downscaled relative to those in WT mice, suggesting that synaptic weakening may reflect an adaptation to aberrant activity. Our observations suggest that cellular and synaptic abnormalities in the AD model represent a maladaptive transformation of the baseline physiological state seen in WT conditions rather than entirely novel and unrelated manifestations.

neuroscience↗

bioRxiv2022

Augmenting TCR signal strength and ICOS costimulation results in metabolically fit and therapeutically potent human CAR Th17 cell therapy

Adoptive cell transfer (ACT) therapy with IL-17 producing human T cells elicits potent antitumor activity in preclinical models. However, further refinement of this novel approach is needed to position it for clinical application. While activation signal strength differentially regulates IL-17 production by human CD4+ T cells, the degree to which TCR and co-stimulation signal strength impacts antitumor Th17 cell immunity remains poorly understood. We discovered that decreasing TCR/co-stimulation signal strength by incremental reduction of CD3/co-stimulation beads in a Th17 culture progressively diminished their effector memory phenotype but enhanced their polyfunctionality. Additional investigation revealed that Th17 cells stimulated with CD3/ICOS beads produced more IL-17A, IFN{gamma}, IL-2 and IL-22 than those stimulated with CD3/CD28 beads, regardless of signal strength. Th17 cells propagated with 30-fold fewer CD3/ICOS beads (weak signal strength, 1 bead per 10 T cells) were less reliant on glucose for growth compared to those stimulated with the standard, strong signal strength (3 beads per T cell). Further metabolomic analysis revealed Th17 cells weakly simulated with CD3/ICOS beads favored the central carbon pathway through increased gluconeogenesis for bioenergetics, marked by abundant intracellular phosphoenoylpyruvate (PEP). Importantly, Th17 cells weakly stimulated with CD3/ICOS beads and redirected with a chimeric antigen receptor (CAR) that recognizes mesothelin were more effective at clearing large human mesothelioma tumors when infused into mice than those manufactured using the standard FDA-approved protocols. Taken together, these data indicate Th17 ACT therapy can be improved by using fewer activation beads during T cell manufacturing, a finding that is both cost effective and directly translatable to patients.

immunology↗

bioRxiv2021

B cells imprint adoptively transferred CD8+ T cells with enhanced tumor immunity

Here we report a novel strategy to reverse the tolerant state of adoptively transferred CD8+ T cells against melanoma through ex vivo expansion with the TLR9 agonist CpG. T cells generated in the presence of CpG display potent anti-tumor efficacy without in vivo co-administration of high dose IL-2 or vaccination, which are classically required for effective treatment of solid tumors using adoptive cell therapies. CD8+ T cells adopt a unique proteomic signature and are characterized by an IL-2RhighICOShighCD39low phenotype after CpG-mediated expansion. Surprisingly, we found that the presence of B cells, in the culture, was essential for imprinting CD8+ T cells with this phenotype and moreover purified B cells were sufficient to mediate the CpG-associated changes in T cells. These findings reveal a vital role for B cells in the generation of effective antitumor CD8+ T cells and have immediate implications for profoundly improving immunotherapy for patients. SUMMARY STATEMENTThe TLR9 agonist CpG allows B cells to license adoptively transferred CD8+ T cells with potent tumor immunity. These licensed T cells have a unique proteomic signature, are marked by low CD39 and high ICOS and IL-2R expression, and engraft robustly in vivo.

immunology↗

bioRxiv2021

Modeling ex vivo tumor-infiltrating lymphocyte expansion from established solid malignancies

Adoptive transfer of tumor-infiltrating lymphocytes (TIL) elicits the regression of metastatic malignancies, yet a low proportion of patients achieve complete durable responses. The high incidence of relapse in these patients highlights the need to better understand mechanisms of tumor escape from T cell control. While melanoma has provided the foundation for developing TIL therapy, much less is known about TIL efficacy and relapse in other malignancies. We sought to investigate TIL characteristics in mouse tumors which have not been studied in this setting. Here, we expanded murine TIL ex vivo in IL-2 from fragments of multiple tumor models, including oral cavity cancer models of varying immunogenicity. Additionally, TIL was expanded from pmel-1 mice bearing B16 melanoma, yielding an enriched population of tumor-infiltrating TCR transgenic T cells. Murine TILs are similar to human TIL in that they express high levels of inhibitory receptors (PD-1, Tim-3, etc.) and can be expanded ex vivo in IL-2 extensively. Of clinical relevance, we draw parallels between murine and patient TIL, evaluating relationships between PD-1, Lag-3, and Tim-3 on TILs from a cohort of oral cavity cancer patients. This platform can be used by labs even in the absence of clinical specimens or clean cell facilities and will be important to more broadly understand TIL phenotypes across many different malignancies.

immunology↗

bioRxiv2020

HIV antiretrovirals have differential effects on viability and function in human iPSC-derived neurons and neural precursor cells

Despite viral suppression due to combination antiretroviral therapy (cART), HIV-associated neurocognitive disorders (HAND) continue to affect half of people with HIV, suggesting that certain antiretrovirals (ARVs) may contribute to HAND. We examined the effects of nucleoside/nucleotide reverse transcriptase inhibitors tenofovir disproxil fumarate (TDF) and emtricitabine (FTC) and the integrase inhibitors dolutegravir (DTG) and elvitegravir (EVG) on viability, structure, and function of glutamatergic neurons (a subtype of CNS neuron involved in cognition) derived from human induced pluripotent stem cells (hiPSC-neurons), and primary human neural precursor cells (hNPCs), which are responsible for neurogenesis. Using automated digital microscopy and image analysis (high content analysis, HCA), we found that DTG, EVG, and TDF decreased hiPSC-neuron viability, neurites, and synapses after seven days of treatment. Analysis of hiPSC-neuron calcium activity using Kinetic Image Cytometry (KIC) demonstrated that DTG and EVG also decreased the frequency and magnitude of intracellular calcium transients. Longer ARV exposures and simultaneous exposure to multiple ARVs increased the magnitude of these neurotoxic effects. Using the Microscopic Imaging of Epigenetic Landscapes (MIEL) assay, we found that TDF decreased hNPC viability and changed the distribution of histone modifications that regulate chromatin packing, suggesting that TDF may reduce neuroprogenitor pools important for CNS development and maintenance of cognition in adults. This study establishes human preclinical assays that can screen potential ARVs for CNS toxicity to develop safer cART regimens and HAND therapeutics.

neuroscience↗

bioRxiv2020

Recognition memory via repetition suppression in mouse hippocampal dorsal CA2 pyramidal neurons expressing the vasopressin 1b receptor

Recognition memory, often compromised in psychiatric disorders, is a major component of declarative memory, which permits the realization that an event, object or social subject has been previously encountered. The CA2 region of the dorsal hippocampus (dCA2) is involved in social memory and responds to novel objects, in time and space. However, it remains unclear how these neurons encode either social or inanimate object recognition. Here, we show that in dCA2, encoding of social recognition memory entails suppression of pyramidal neurons activity leading to a sparse representation of the familiar conspecific. We discuss the neural coding scheme by which dCA2 pyramidal neurons contribute to social memory.

neuroscience↗

bioRxiv2019

Regulation of defeat-induced social avoidance by medial amygdala DRD1 in male and female prairie voles

Social interaction with unfamiliar individuals is necessary for species-preserving behaviors such as finding mates and establishing social groups. However, social conflict is a potential negative outcome to interaction with a stranger that can be distressing enough to cause an individual to later avoid interactions with other unfamiliar conspecifics. Unfortunately, stress research using a prominent model of social conflict, social defeat stress, has largely omitted female subjects. This has left a void in the literature regarding social strain on female stress biology and adequate comparison of the effect of sex in stress pathways. The prairie vole (Microtus ochrogaster) exhibits aggressive behavior in both sexes, making voles an attractive candidate to model social defeat in both sexes. This study sought to establish a model of social defeat stress in both male and female prairie voles, characterize behavioral changes in response to this stressor, and investigate the role of dopamine signaling in the response to social defeat stress. Defeated male and female prairie voles displayed social avoidance as well as an increase in expression of dopamine receptor D1 (DRD1) in the medial amygdala (MeA). Pharmacological manipulation of DRD1 signaling in the MeA revealed that increased DRD1 signaling is sufficient to induce a social avoidant state, and could be a necessary component in the defeat-induced social avoidance response. These findings provide the prairie vole as a model of social defeat in both sexes, and implicate the MeA in avoidance of unfamiliar conspecifics after a distressing social encounter.

neuroscience↗

bioRxiv2019

Chromatin compartment dynamics in a haploinsufficient model of cardiac laminopathy

Pathogenic mutations in A-type nuclear lamins cause dilated cardiomyopathy, which is postulated to result from dysregulated gene expression due to changes in chromatin organization into active and inactive compartments. To test this, we performed genome-wide chromosome conformation analyses (Hi-C) in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) with a haploinsufficient mutation for lamin A/C. Compared to gene-corrected cells, mutant hiPSC-CMs have marked electrophysiological and contractile alterations, with modest gene expression changes. While large-scale changes in chromosomal topology are evident, differences in chromatin compartmentalization are limited to a few hotspots that escape inactivation during cardiogenesis. These regions exhibit upregulation of multiple non-cardiac genes including CACNA1A, encoding for neuronal P/Q-type calcium channels. Pharmacological inhibition of the resulting current partially mitigates the electrical alterations. On the other hand, A/B compartment changes do not explain most gene expression alterations in mutant hiPSC-CMs. We conclude that global errors in chromosomal compartmentation are not the primary pathogenic mechanism in heart failure due to lamin A/C haploinsufficiency. SummaryBertero et al. observe that lamin A/C haploinsufficiency in human cardiomyocytes markedly alters electrophysiology, contractility, gene expression, and chromosomal topology. Contrary to expectations, however, changes in chromatin compartments involve just few regions, and most dysregulated genes lie outside these hotspots. Condensed titleGenomic effects of lamin A/C haploinsufficiency

genomics↗

bioRxiv2017

Myosin IIA interacts with the spectrin-actin membrane skeleton to control red blood cell membrane curvature and deformability

The biconcave disc shape and deformability of mammalian red blood cells (RBCs) relies upon the membrane skeleton, a viscoelastic network of short, membrane-associated actin filaments (F-actin) cross-linked by long, flexible spectrin tetramers. Nonmuscle myosin II (NMII) motors exert force on diverse F-actin networks to control cell shapes, but a function for NMII contractility in the 2D spectrin-F-actin network in RBCs has not been tested. Here, we show that RBCs contain membrane skeleton-associated NMIIA puncta, identified as bipolar filaments by super-resolution fluorescence microscopy. NMIIA association with the membrane skeleton is ATP-dependent, consistent with NMIIA motor domains binding to membrane skeleton F-actin and contributing to membrane mechanical stability. In addition, the NMIIA heavy and light chains are phosphorylated in vivo in RBCs, indicating active regulation of NMIIA motor activity and filament assembly, while reduced heavy chain phosphorylation of membrane skeleton-associated NMIIA indicates assembly of stable filaments at the membrane. Treatment of RBCs with blebbistatin, an inhibitor of NMII motor activity, decreases the number of NMIIA filaments associated with the membrane and enhances local, nanoscale membrane oscillations, suggesting decreased membrane tension. Blebbistatin-treated RBCs also exhibit elongated shapes, loss of membrane curvature, and enhanced deformability, indicating a role for NMIIA contractility in promoting membrane stiffness and maintaining RBC biconcave disc cell shape. As structures similar to the RBC membrane skeleton are conserved in many metazoan cell types, these data demonstrate a general function for NMII in controlling specialized membrane morphology and mechanical properties through contractile interactions with short F-actin in spectrin-F-actin networks.\n\nSignificance statementThe biconcave disc shape and deformability of the mammalian RBC is vital to its circulatory function, relying upon a 2D viscoelastic spectrin-F-actin network attached to the membrane. A role for myosin II (NMII) contractility in generating tension in this network and controlling RBC shape has never been tested. We show that NMIIA forms phosphorylated bipolar filaments in RBCs, which associate with F-actin at the membrane. NMIIA motor activity is required for interactions with the spectrin-F-actin network, and regulates RBC biconcave shape and deformability. These results provide a novel mechanism for actomyosin force generation at the plasma membrane, and may be applicable to other cell types such as neurons and polarized epithelial cells with a spectrin-F-actin-based membrane skeleton.

cell biology↗

bioRxiv2017

PI3Kδ inhibition supports memory T cells with enhanced antitumor fitness

Phosphatidylinositol-3-kinase p110{delta} (PI3K{delta}) inhibition by Idelalisib (CAL-101) in hematological malignancies directly induces apoptosis in cancer cells and disrupts immunological tolerance by depleting regulatory T cells (Tregs). Yet, little is known about the direct impact of PI3K{delta} blockade on effector T cells from CAL-101 therapy. Herein, we demonstrate a direct effect of p110{delta} inactivation via CAL-101 on murine and human CD8+ T cells that promotes a strong undifferentiated memory phenotype (elevated CD62L/CCR7, CD127 and Tcf7). These CAL-101 T cells also persisted longer after transfer and exerted stronger antitumor immunity compared to traditionally expanded CD8+ T cells in two solid tumor models. Thus, this report describes a novel direct enhancement of CD8+ T cell memory by a p110{delta} inhibitor that leads to markedly improved tumor regression. This finding has significant implications to improve outcomes from next generation cancer immunotherapies.\n\nHighlightsO_LIIn vitro blockade of PI3K p110{delta} with CAL-101 endows antitumor T cells with a stronger memory phenotype than those treated with AKTi\nC_LIO_LIThe strong memory phenotype of CAL-101 treated cells translates into improved survival of mice bearing aggressive tumors after adoptive transfer of these T cells\nC_LIO_LIHuman CAR engineered T cells treated with CAL-101 possess an enhanced memory phenotype and robust antitumor efficacy\nC_LIO_LIThe antitumor efficacy of CAL-101 primed T cells is not mediated by high CD62L or CD127 expression, but is likely driven by their stem memory phenotype\nC_LI\n\neTOC BlurbBowers et al report a novel function of PI3K blockade using the p110{delta} subunit inhibitor CAL-101 to induce memory and antitumor potency in CD8+ T cells. Ex vivo treatment of T cells with CAL-101 leads to improved antitumor control and subject survival in both murine transgenic T cell and human CAR T cell models.

cancer biology↗