Biology subjects

Howe, C. L.

Publications and source records attributed to Howe, C. L..

10 recordsLinked to original sources

bioRxiv2026

Human neurons stimulated with IFNγ present HLA class I-restricted autoantigens to cytotoxic CD8+ T cells

Interferon-{gamma} (IFN{gamma}) signaling is prominent in inflammatory CNS microenvironments across many neurological disorders, but the neuronal peptides presented on HLA class I under these conditions and their functional consequences for CD8+ T cells remain incompletely defined. Here we combine human iPSC-derived human neural aggregates (HNAs), HLA class I immunoprecipitation coupled to LC-MS/MS immunopeptidomics, and microfluidic co-culture assays to map IFN{gamma}-induced neuronal antigen presentation and test antigen-specific cytotoxicity. IFN{gamma} stimulation induced robust HLA class I expression in HNAs and enabled recovery of a canonical 8-12-mer class I ligandome enriched for 9-mers. Neuron-restricted expression of a synapsin-driven polyepitope cassette yielded presentation of defined neoantigen 9-mer peptides on donor HLA class I molecules and, in the presence of IFN{gamma}, elicited activation of autologous antigen-specific CD8+ T cells and consequent antigen-dependent neurite injury. Across four donors, comparative immunopeptidomics identified large IFN{gamma}-unique neural peptide repertoires distinct from matched fibroblasts and revealed a consistent enrichment of predicted high-affinity binders on HLA-B allotypes. Finally, {beta}2-microglobulin deletion ablated peptide recovery, and neuron-restricted {beta}2-microglobulin reconstitution enabled identification of neuron-derived peptides, including peptides derived from neurofilament light (NEFL) that were shared across donors and presented on multiple HLA allotypes. Together, these data provide an integrated platform for neuronal autoantigen discovery and functional validation and support a model in which IFN{gamma}-driven neuronal HLA class I presentation creates an HLA-B-weighted epitope landscape that can be recognized by autoreactive cytotoxic CD8+ T cells.

immunology↗

bioRxiv2026

Characterization of HLA-restricted GAD65-specific CD8+ T cell responses in patients with GAD65 antibody-associated neurological disorders

BackgroundHigh-titer glutamic acid decarboxylase 65 (GAD65) antibodies are found in patients with GAD65 antibody-associated neurological disorders, including stiff-person syndrome (SPS), GAD65 cerebellar ataxia (CA), and GAD65 epilepsy. Given the intracellular localization of the antigenic target, a direct pathogenic role for GAD65 autoantibodies is unlikely. Instead, the autoantibody may be a biomarker for the existence of pathogenic anti-GAD65 autoreactive CD8+ T cells. MethodsPBMC-derived dendritic cells (DCs) from 20 GAD65 antibody-positive patients with neurological manifestations (SPS, n=10; CA, n=7; epilepsy, n=3) and 15 healthy controls were pulsed with full-length GAD65 protein, full-length GAD67 protein, overlapping 15-mer peptide pools spanning GAD65 and GAD67, individual GAD65 15-mer peptides, or GAD65 9-mer peptides selected from predicted class I binding hotspots. T cell activation was quantified by flow cytometry-based activation-induced marker (AIM) assays using CD69 upregulation. Class I and class II HLA haplotypes were determined by high resolution typing. NetMHCpan v4.2c was used to generate residue-level peptide:HLA binding density maps across GAD65, and candidate 9-mers were validated for HLA binding by peptide:MHC monomer affinity testing. GAD65-peptide-HLA-restricted CD8+ T cells were identified by co-staining with two separately assembled tetramers carrying the same peptide:HLA complex on different fluorophores (APC and BV421), with double-positive events scored as antigen-specific. HLA-restricted cytotoxicity was measured by coculture of patient CD8+ T cells with GAD65-expressing HEK-293T cells reconstituted with defined HLA class I alleles using AAV-delivered Cre-dependent HLA-2A-eGFP cassettes. ResultsCD8+ T cells from GAD65 antibody-positive patients showed increased activation in response to DCs pulsed with full-length GAD65 relative to healthy controls (P=0.0157, Welchs t-test), whereas responses to GAD67 did not differ significantly between groups. CD4+ T cells responded to both GAD65 (P=0.0004) and GAD67 (P=0.0051). Peptide pool screening of GAD65 identified discrete CD8+ and CD4+ immunogenic regions, with refinement using individual 15-mers localizing CD8+ activity to multiple subdomains within GAD65(205-300), GAD65(316-435), and GAD65(447-520). HLA class I haplotyping in 16 non-Hispanic White GAD65+ patients revealed enrichment of HLA-B*08:01 (3.0-fold) and HLA-B*40:01 (4.1-fold) relative to USA NMDP European Caucasian reference frequencies (both BH q < 0.05), with suggestive enrichment of HLA-C*03:04 (2.9-fold; q = 0.09). Additionally, 44% of patients carried the HLA-A*01:01, HLA-B*08:01, and HLA-C*07:01 8.1 ancestral haplotype, approximately four-fold higher than the expected population frequency. Dual-fluorophore tetramer staining identified CD8+ T cells in GAD65+ subjects that bound a subset of HLA-A*11:01- and HLA-B*08:01-restricted GAD65 9-mers, with the clearest disease-skewed signals localized to GAD65(213-221), GAD65(257-265), and GAD65(529-537). In an HLA-reconstituted target-cell killing assay, CD8+ T cells from an HLA-B*08:01-positive GAD65+ patient mediated antigen- and HLA-restricted depletion of GAD65-expressing HEK-293T cells, with HLA-restricted target loss also observed in single-donor experiments across additional HLA-A, -B, and -C contexts. ConclusionsPatients with GAD65 antibody-associated neurological disorders harbor circulating CD8+ T cells that recognize discrete HLA class I-restricted GAD65 peptides and that are capable of cytotoxicity against GAD65-expressing HLA-matched target cells. We characterize the immunogenetic and cellular features of class I-restricted CD8+ T cell responses in GAD65 autoimmunity, including overrepresentation of the 8.1 ancestral haplotype, complementing the historical focus on antibodies and CD4+ T cell help, and we provide a panel of validated GAD65 peptide:HLA tetramers for prospective isolation, clonotypic analysis, and longitudinal monitoring of candidate pathogenic CD8+ T cell populations across the GAD65 antibody-associated neurological disease spectrum.

immunology↗

bioRxiv2025

Light-field deep learning enables high-throughput, scattering-mitigated calcium imaging

Light field microscopy (LFM) enables volumetric, high throughput functional imaging. However, the computational burden and vulnerability to scattering limit LFMs application to neuroscience. We present a light-field strategy for volumetric, scattering-mitigated neural circuit activity monitoring. A physics-based deep neural network, LNet, is trained with two-photon volumes and one-photon light fields. A processing pipeline uses LNet to extract calcium activity from light-field videos of jGCaMP8f-expressing neurons in acute cortical slices. The extracted time series have high signal-to-noise ratios and reduced optical crosstalk compared to conventional volume reconstruction methods. Imaging 100 volumes per second, we observe putative spikes fired at up to 10 Hz and the spatial intermingling of putative ensembles throughout 530 x 530 x 100-micron volumes. Compared to iterative algorithms, LNet workflows reduce light-field video processing times by 2- to 12-fold, advancing the goal of real-time, scattering-robust volumetric neural circuit imaging for closed-loop and adaptive experimental paradigms.

bioengineering↗

bioRxiv2022

Physics-based Deep Learning for Imaging Neuronal Activity via Two-photon and Light Field Microscopy

Light Field Microscopy (LFM) is an imaging technique that offers the opportunity to study fast dynamics in biological systems due to its rapid 3D imaging rate. In particular, it is attractive to analyze neuronal activity in the brain. Unlike scanning-based imaging methods, LFM simultaneously encodes the spatial and angular information of light in a single snapshot. However, LFM is limited by a trade-off between spatial and angular resolution and is affected by scattering at deep layers in the brain tissue. In contrast, two-photon (2P) microscopy is a point-scanning 3D imaging technique that achieves higher spatial resolution, deeper tissue penetration, and reduced scattering effects. However, point-scanning acquisition limits the imaging speed in 2P microscopy and cannot be used to simultaneously monitor the activity of a large population of neurons. This work introduces a physics-driven deep neural network to image neuronal activity in scattering volume tissues using LFM. The architecture of the network is obtained by unfolding the ISTA algorithm and is based on the observation that the neurons in the tissue are sparse. The deep-network architecture is also based on a novel imaging system modeling that uses a linear convolutional neural network and fits the physics of the acquisition process. To achieve the high-quality reconstruction of neuronal activity in 3D brain tissues from temporal sequences of light field (LF) images, we train the network in a semi-supervised manner using generative adversarial networks (GANs). We use the TdTomato indicator to obtain static structural information of the tissue with the microscope operating in 2P scanning modality, representing the target reconstruction quality. We also use additional functional data in LF modality with GCaMP indicators to train the network. Our approach is tested under adverse conditions: limited training data, background noise, and scattering samples. We experimentally show that our method performs better than model-based reconstruction strategies and typical artificial neural networks for imaging neuronal activity in mammalian brain tissue, considering reconstruction quality, generalization to functional imaging, and reconstruction speed.

neuroscience↗

bioRxiv2020

Comparing volumetric reconstruction algorithms for light field imaging of high signal-to-noise ratio neuronal calcium transients

SignificanceLight-field microscopy (LFM) enables fast, light-efficient, volumetric imaging of neuronal activity with calcium indicators. Calcium transients differ in temporal signal-to-noise ratio (tSNR) and spatial confinement when extracted from volumes reconstructed by different algorithms. AimWe evaluated the capabilities and limitations of two light-field reconstruction algorithms for calcium fluorescence imaging. ApproachWe acquired light-field image series from neurons either bulk-labeled or filled intracellularly with the red-emitting calcium dye CaSiR-1 in acute mouse brain slices. We compared the tSNR and spatial confinement of calcium signals extracted from volumes reconstructed with synthetic refocusing and Richardson-Lucy 3D deconvolution with and without total variation regularization. ResultsBoth synthetic refocusing and Richardson-Lucy deconvolution resolved calcium signals from single cells and neuronal dendrites in three dimensions. Increasing deconvolution iteration number improved spatial confinement but reduced tSNR compared to synthetic refocusing. Volumetric light-field imaging did not decrease calcium signal tSNR compared to interleaved, widefield image series acquired in matched planes. ConclusionsLFM enables high-volume rate, volumetric imaging of calcium transients in single cells (bulk-labeled), somata and dendrites (intracellular loaded). The trade-offs identified for tSNR, spatial confinement, and computational cost indicate which of synthetic refocusing or deconvolution can better realize the scientific requirements of future LFM calcium imaging applications.

bioengineering↗

bioRxiv2020

Subcellular resolution 3D light field imaging with genetically encoded voltage indicators

Light field microscopy (LFM) enables high signal-to-noise ratio (SNR), light efficient volume imaging at fast frame rates, and has been successfully applied to single-cell resolution functional neuronal calcium imaging. Voltage imaging with genetically encoded voltage indicators (GEVIs) stands to particularly benefit from light field microscopys volumetric imaging capability due to high required sampling rates, and limited probe brightness and functional sensitivity. Previous LFM studies have imaged GEVIs to track population-level interactions only in invertebrate preparations and without single cell resolution. Here we demonstrate sub-cellular resolution GEVI light field imaging in acute mouse brain slices resolving dendritic voltage signals localized in three dimensions. We characterize the effects of different light field reconstruction techniques on the SNR and signal localization and compare the SNR to fluorescence transients imaged in wide field. Our results demonstrate the potential of light field voltage imaging for studying dendritic integration and action potential propagation and backpropagation in 3 spatial dimensions.

neuroscience↗

bioRxiv2019

Dedicated setup for the photoconversion of fluorescent dye for functional electron microscopy.

Here, we describe a cost-effective setup for targeted photoconversion of fluorescent signals into electron dense ones. This approach has offered invaluable insights in the morphology and function of fine neuronal structures. The technique relies on the localized oxidation of diaminobenzidine (DAB) mediated by excited fluorophores. This paper includes a detailed description of how to build a simple photoconversion setup that can increase reliability and throughput of this well-established technique. The system described here, is particularly well-suited for thick neuronal tissue, where light penetration and oxygen diffusion may be limiting DAB oxidation. To demonstrate the system, we use Correlative Light and Electron Microscopy (CLEM) to visualize functionally-labelled individual synaptic vesicles released onto an identified layer 5 neuron in an acute cortical slice. The setup significantly simplifies the photoconversion workflow, increasing the depth of photoillumination, improving the targeting of the region of interest and reducing the time required to process each individual samples. We have tested this setup extensively for the photoconversion of FM 1-43FX and Lucifer Yellow both excited at 473 nm. In principle, the system can be adapted to any dye or nanoparticle able to oxidize DAB when excited by a specific light wavelength.

neuroscience↗

bioRxiv2018

Surface Plasmon Resonance Imaging of Excitable Cells

Surface plasmons are highly sensitive to refractive index variations adjacent to the surface. This sensitivity has been exploited successfully for chemical and biological assays. In these systems, a surface plasmon resonance (SPR)-based sensor detects temporal variations in the refractive index at a point. SPR has also been used in imaging systems where the spatial variations of refractive index in the sample provide the contrast mechanism. A high numerical aperture objective lens has been used to design SPR microscopy systems with the ability to image adherent live cells. Addressing research questions in cell physiology and pharmacology often requires the development of a multimodal microscope where complementary information can be obtained.\n\nIn this paper, we present the development of a multimodal microscope that combines surface plasmon resonance imaging with a number of additional imaging modalities including bright-field, epi-fluorescence, total internal reflection microscopy (TIRM) and SPR fluorescence microscopy. We used a high numerical aperture objective lens to achieve SPR and TIR microscopy with the ability to image adherent live cells non-invasively. The platform has been used to image live cell cultures demonstrating both fluorescent and label-free techniques. The SPR and TIR imaging systems feature a wide field of view (300 {micro}m) that allows measurements from multiple cells while the resolution is sufficient to image fine cellular processes. The ability of the platform to perform label-free functional imaging of living cell was demonstrated by imaging the spatial variations in contraction of stem cell-derived cardiomyocytes. This technique has a promise for non-invasive imaging of the development of cultured cells over very long periods of time.

bioengineering↗

bioRxiv2017

Neuronal CCL2 expression drives inflammatory monocyte infiltration into the brain during acute virus infection

BackgroundViral encephalitis is a dangerous compromise between the need to robustly clear pathogen from the brain and the need to protect neurons from bystander injury. Theiler's murine encephalomyelitis virus (TMEV) infection of C57Bl/6 mice is a model of viral encephalitis in which the compromise results in hippocampal damage and permanent neurological sequelae. We previously identified brain infiltrating inflammatory monocytes as the primary driver of this hippocampal pathology, but the mechanisms involved in recruiting these cells to the brain were unclear.\n\nMethodsChemokine expression levels in the hippocampus were assessed by microarray, ELISA, RT-PCR, and immunofluorescence. Monocyte infiltration during acute TMEV infection was measured by flow cytometry. CCL2 levels were manipulated by immunodepletion and by specific removal from neurons in mice generated by crossing a line expressing the Cre recombinase behind the synapsin promoter to animals with floxed CCL2.\n\nResultsInoculation of the brain with TMEV induced hippocampal production of the proinflammatory chemokine CCL2 that peaked at 6 hours postinfection, whereas inoculation with UV-inactivated TMEV did not elicit this response. Immunofluorescence revealed that hippocampal neurons expressed high levels of CCL2 at this timepoint. Genetic deletion of CCR2 and systemic immunodepletion of CCL2 abrogated or blunted the infiltration of inflammatory monocytes into the brain during acute infection. Specific genetic deletion of CCL2 from neurons reduced serum and hippocampal CCL2 levels and inhibited inflammatory monocyte infiltration into the brain.\n\nConclusionsWe conclude that intracranial inoculation with infectious TMEV rapidly induces the expression of CCL2 in neurons, and this cellular source is necessary for CCR2-dependent infiltration of inflammatory monocytes into the brain during the most acute stage of encephalitis. These findings highlight a unique role for neuronal production of chemokines in the initiation of leukocytic infiltration into the infected central nervous system.

neuroscience↗

bioRxiv2017

Systemic evidence of acute seizure-associated neuronal injury in patients with temporal lobe epilepsy

Patients with drug refractory temporal lobe epilepsy frequently accumulate cognitive impairment over time, suggesting neuronal loss induced by seizures. We measured serum levels of neuron-specific enolase (NSE), a neuronal injury marker, relative to levels of S100{beta}, a marker of glial injury, at 6 AM, 9 AM, noon, 3 PM, and 6 PM over the course of several days in 7 epilepsy patients and 4 healthy controls. All epilepsy patients exhibited significant deviations in NSE levels through time, and 4 of the epilepsy patients exhibited large sample entropy values and large signal variation metrics for NSE relative to S100{beta}. Controls did not exhibit such changes. Correlation analysis revealed that NSE levels were significantly elevated after clinical seizure events. There was also a highly significant relationship between increased EEG spike frequency and an increase in serum NSE levels measured 24 hours later. The detection of large but transient post-ictal increases in NSE suggests that even self-limited seizures may cause an injury to neurons that underlies cognitive decline in some patients. Post-ictal assessment of serum NSE may serve as a biomarker for measuring the efficacy of future acute neuroprotective strategies in epilepsy patients.

neuroscience↗