Biology subjects

Smith, B. E.

Publications and source records attributed to Smith, B. E..

10 recordsLinked to original sources

bioRxiv2026

Activation-dependent lentiviruses enable antigen-specific T cell expansion and transduction

Cancer immunotherapies rely on tumor-specific T cells, which arise endogenously in most patients with cancer, but can be low frequency and poorly functional. Methods to specifically identify, expand, and manipulate tumor-specific T cells at the rare frequencies found in peripheral blood would enable new immunotherapeutic strategies. Here, we demonstrate an approach to virally transduce polyclonal tumor-reactive T cells across any MHC haplotype and in the absence of knowing the cognate antigen. By generating lentiviral vectors that selectively transduce cells expressing 4-1BB (CD137), a marker of T cell activation, we can transduce antigen-specific T cells with user-defined genetic cargoes that can selectively expand and track individual clonotypes via single-cell sequencing. Anti-4-1BB lentiviruses (4-1BB LVs) encoding therapeutic cargoes can also enhance antigen-specific T cells to extend survival in a xenograft model of human melanoma and transduce tumor-infiltrating T cells from patients with ovarian cancer. Overall, the 4-1BB LV platform targets antigen-specific T cells in a manner agnostic to both the antigen and presenting MHC, with potential applications in adoptive cell therapy manufacturing and TCR identification. One Sentence SummaryEngineered lentiviral vectors targeting 4-1BB selectively activate, expand, and transduce antigen-specific T cells with immunomodulatory cargo.

immunology↗

bioRxiv2026

Mueller glia-vasculature interactions in the developing retina

Coordinated signaling among neurons, glia, and the vasculature is essential for the formation of a functional nervous system, yet how these relationships emerge during development remains unclear. Here, we investigated the developmental interplay between neural activity, Muller glia, and the retina vasculature in mice. Using quantitative confocal imaging from postnatal day 5 to eye-opening, we mapped the emergence of the superficial, intermediate, and deep vasculature layers and found that they emerged normally in mice lacking the {beta}2-containing nicotinic acetylcholine receptors, despite a dramatic reduction in cholinergic signaling. Tip cell density and overall vessel growth were unchanged, indicating cholinergic wave activity is not required for the emergence of retinal vasculature. We next defined the developmental timeline of Muller glia-vascular interactions. Sparse labeling and immunohistochemistry revealed that Muller glial lateral processes closely associate with endothelial tip cells during intermediate- and deep-layer angiogenesis and establish Aquaporin-4-enriched endfeet at vascular contact sites from the earliest stages of growth, even when vessel trajectories are perturbed. Finally, two-photon calcium imaging combined with simultaneous electrophysiology demonstrated that Muller glial endfeet exhibit robust, compartmentalized calcium transients during development. Although a subset of events was temporally correlated with retinal waves, enhancing neurotransmitter spillover selectively increased wave-associated activity in glial stalks but not endfeet. These findings indicate that calcium signaling at the glial-vascular interface is largely independent of spontaneous neuronal activity. Together, our results support a model in which Muller glia engage growing vessels through an activity-independent, parallel developmental program that may provide instructive cues for retinal angiogenesis.

neuroscience↗

bioRxiv2025

Scalable TCR synthesis and screening enables antigen reactivity mapping in vitiligo

T cell receptors (TCRs) mediate antigen recognition in adaptive immunity, yet large-scale mapping of TCR-antigen interactions remains a major challenge. Current approaches to synthesize and functionally screen TCRs remain technically complex and limited in throughput. We introduce a modular strategy, TCRAFT, to rapidly construct tens of thousands of TCRs for <$1 each while maintaining TCR-{beta} pairing with >99% accuracy. We integrate this approach with a high-throughput antigen discovery platform to enable library-on-library TCR-antigen screening. We reconstruct and screen 3,808 TCRs from vitiligo lesions, linking TCR specificity to transcriptional phenotypes for antigen-reactive T cells. To demonstrate scalability, we synthesize and screen 30,810 TCRs from donors with pancreatic ductal adenocarcinoma to capture antigen-specific TCRs. This workflow reduces the cost and complexity of large-scale TCR screening, enabling the expansion of the known landscape of antigen-specific TCRs in vitiligo with a method that can be readily extended to other immunological applications.

immunology↗

bioRxiv2024

The evolution of weaponry and aggressive behaviour in field crickets

Weapons are among the most extravagant sexually selected traits known, yet the evolution of weapon diversity remains understudied. We used field crickets (Orthoptera, Gryllinae) to test two hypotheses explaining interspecific diversity in weaponry. We raised eight species of Gryllus field crickets under common garden conditions and staged interactions between conspecific males. We measured body size and weapon shape (relative head and mouthpart size) to determine weapon allometry in both males and females, quantified the intensity of male-male aggression for each species, analyzed the effects of both body size and weapon shape on contest outcome, and tested comparative relationships between morphology and behaviour using phylogenetic least squares regression. We found that larger males won more contests than smaller males in seven of eight species, and weapon shape predicted contest success in only one species. Contrary to the fighting advantage hypothesis, body size was not related to aggressiveness across species, but weaponry was. Additionally, the most aggressive species had the most elaborate weaponry, contrary to the weapon-signal continuum hypothesis. Our results highlight the complexity of weaponry evolution in a group of organisms that has been a model system for the observation and study of aggression for approximately 1000 years.

evolutionary biology↗

bioRxiv2024

Peptide-MHC-targeted retroviruses enable in vivo expansion and gene delivery to tumor-specific T cells

Tumor-infiltrating-lymphocyte (TIL) therapy has demonstrated that endogenous T cells can be harnessed to initiate an effective anti-tumor response. Despite clinical promise, current TIL production protocols involve weeks-long ex vivo expansions which can affect treatment efficacy. Therefore, additional tools are needed to engineer endogenous tumor-specific T cells to have increased potency while mitigating challenges of manufacturing. Here, we present a strategy for pseudotyping retroviral vectors with peptide-major histocompatibility complexes (pMHC) for antigen-specific gene delivery to CD8 T cells and examine the efficacy of these transduced cells in immunocompetent mouse models. We demonstrate that pMHC-targeted viruses are able to specifically deliver function-enhancing cargoes while simultaneously activating and expanding anti-tumor T cells. The specificity of these viral vectors enables in vivo engineering of tumor-specific T cells, circumventing ex vivo manufacturing processes and improving overall survival in B16F10-bearing mice. Altogether, we have established that pMHC-targeted viruses are efficient vectors for reprogramming and expanding tumor-specific populations of T cells directly in vivo, with the potential to substantially streamline engineered cell therapy production for a variety of applications.

immunology↗

bioRxiv2024

Library-based single-cell analysis of CAR signaling reveals drivers of in vivo persistence

The anti-tumor function of engineered T cells expressing chimeric antigen receptors (CARs) is dependent on signals transduced through intracellular signaling domains (ICDs). Different ICDs are known to drive distinct phenotypes, but systematic investigations into how ICD architectures direct T cell function--particularly at the molecular level--are lacking. Here, we use single-cell sequencing to map diverse signaling inputs to transcriptional outputs, focusing on a defined library of clinically relevant ICD architectures. Informed by these observations, we functionally characterize transcriptionally distinct ICD variants across various contexts to build comprehensive maps from ICD composition to phenotypic output. We identify a unique tonic signaling signature associated with a subset of ICD architectures that drives durable in vivo persistence and efficacy in liquid, but not solid, tumors. Our findings work toward decoding CAR signaling design principles, with implications for the rational design of next-generation ICD architectures optimized for in vivo function.

immunology↗

bioRxiv2023

Pneumococcal Extracellular Vesicles Mediate Horizontal Gene Transfer via the Transformation Machinery

Bacterial cells secrete extracellular vesicles (EVs), the function of which is a matter of intense investigation. Here, we show that the EVs secreted by the human pathogen Streptococcus pneumoniae (pneumococcus) are associated with bacterial DNA on their surface and can deliver this DNA to the transformation machinery of competent cells. These findings suggest that EVs contribute to gene transfer in Gram-positive bacteria, and in doing so, may promote the spread of drug resistance genes in the population. SignificanceThis work extends our understanding of horizontal gene transfer and the roles of extracellular vesicles in pneumococcus. This bacterium serves as the model for transformation, a process by which bacteria can take up naked DNA from the environment. Here we show that extracellular vesicles secreted by the pneumococcus have DNA on their surface, and that this DNA can be imported by the transformation machinery facilitating gene transfer. Understanding EV-mediated gene transfer may provide new avenues to manage the spread of antibiotic drug resistance.

microbiology↗

bioRxiv2022

Cellular Mechanisms Underlying Embryonic Retinal Waves

Spontaneous activity is a hallmark of developing neural systems. In the retina, spontaneous activity comes in the form of retinal waves, comprised of three stages persisting from embryonic day 16 (E16) to eye opening at postnatal day 14 (P14). Though postnatal retinal waves have been well characterized, little is known about the spatiotemporal properties or the mechanisms mediating embryonic retinal waves, designated Stage 1 waves. Using a custom-built macroscope to record spontaneous calcium transients from whole embryonic retinas, we show that Stage 1 waves are initiated at several locations across the retina and propagate across finite regions of a broad range of areas. A gap junction antagonist, meclofenamic acid, reduced the frequency and size of Stage 1 waves but did not abolish them. The general nAChR antagonist, hexamethonium blocked Stage 1 waves, while they persisted in the presence of 4{beta}2 nAChR antagonist dihydro-{beta}-erythroidine, indicating that the spatiotemporal properties of Stage 1 waves are mediated by a complex circuitry involving subtypes of nAChRs and gap junctions. Stage 1 waves in mice lacking the {beta}2 subunit of the nAChRs ({beta}2-nAChR-KO) were reduced, but in contrast to WT mice, they persisted in the hexamethonium and were completely blocked by meclofenamic acid. To assay the impact of Stage 1 waves on retinal development, we compared the spatial distribution of a subtype of retinal ganglion cells, intrinsically photosensitive retinal ganglion cells (ipRGCs) in WT and {beta}2-nAChR-KO mice. We found that the developmental decrease of ipRGC density is preserved between WT and {beta}2-nAChR-KO mice, indicating that processes regulating ipRGC distribution are not influenced by spontaneous activity.

neuroscience↗

bioRxiv2021

Antigen identification and high-throughput interaction mapping by reprogramming viral entry

Deciphering immune recognition is critical for understanding a broad range of diseases and for the development of effective vaccines and immunotherapies. Efforts to do so are limited by a lack of technologies capable of simultaneously capturing the complexity of adaptive immune receptor repertoires and the landscape of potential antigens. To address this, we present RAPTR (Receptor-Antigen Pairing by Targeted Retroviruses). RAPTR combines viral pseudotyping and molecular engineering approaches to enable one-pot library on library interaction screens by displaying antigens on the surface of lentiviruses and encoding their identity in the viral genome. Antigen-specific viral infection of cells allows readout of both antigen and receptor identities via single-cell sequencing. The resulting system is modular, scalable, and compatible with any cell type, making it easily implemented. These techniques provide a suite of new tools for targeted viral entry, molecular engineering, and interaction screens with broad potential applications.

immunology↗

bioRxiv2019

Dynamics of T cell activation mediated by eIF3 interactions with T cell receptor mRNAs

Activation of T cells requires a global surge in cellular protein synthesis, accompanied by a large increase in translation initiation1-4. A central component of the translation initiation machinery-the multi-subunit eukaryotic initiation factor 3 (eIF3)-is rapidly turned on when quiescent T cells are stimulated3. However, the precise role eIF3 plays in activated T cells is not known. Using a global transcriptome crosslinking approach, we show human eIF3 interacts with a distinct set of mRNAs in activated Jurkat cells. A subset of these mRNAs, including those encoding the T cell receptor (TCR) subunits TCRA and TCRB, crosslink to eIF3 across the entire length of the mRNA. The TCRA and TCRB mRNAs do not co-localize with translationally repressed environments of P-bodies or stress granules but form distinct granules, potentially acting as translation "hot-spots." T cell activation through CD28 causes a burst of TCR translation controlled by elements in the 3-untranslated regions (3-UTRs) of the TCRA and TCRB mRNAs that directly contact eIF3 and that are required for T cell activity. These results highlight a new role for eIF3 in regulating the translation dynamics of the TCR and provide insights that can guide the engineering of T cells used in cell immunotherapy applications.

molecular biology↗