Biology subjects

Pichler, B. J.

Publications and source records attributed to Pichler, B. J..

9 recordsLinked to original sources

bioRxiv2024

Making the effect visible - OX40 targeting nanobodies for in vivo imaging of activated T cells

PurposeHuman OX40 (hOX40/CD134), a member of the TNF receptor superfamily, is mainly expressed on activated T lymphocytes. Triggered by its ligand OX40L (CD252), it provides costimulatory signals that support the differentiation, proliferation and long-term survival of T cells. Besides being a relevant therapeutic target, hOX40 is also an important biomarker for monitoring the presence or infiltration of activated T cells within the tumor microenvironment (TME), the inflammatory microenvironment (IME) in immune-mediated diseases (IMIDs) and the lymphatic organs. Here, we developed novel single domain antibodies (nanobodies, Nbs) targeting hOX40 to monitor the activation status of T cells by in vivo molecular imaging. MethodsNbs against hOX40 (hOX40-Nbs) were selected from an immunized Nb-library by phage display. The identified hOX40-Nbs were characterized in vitro, including determination of their specificity, affinity, stability, epitope recognition and their impact on OX40 signaling and T cell function. A lead candidate was site-specifically conjugated with a fluorophore via sortagging and applied for noninvasive in vivo optical imaging (OI) of hOX40-expressing cells in a xenograft mouse model. ResultsOur selection campaign revealed four unique Nbs that exhibit strong binding affinities and high stabilities under physiological conditions. Epitope binning and domain mapping indicated the targeting of at least two different epitopes on hOX40. When analyzing their impact on OX40 signaling, an agonistic effect was excluded for all validated Nbs. Incubation of activated T cells with hOX40-Nbs did not affect cell viability or proliferation patterns, whereas differences in cytokine release were observed. In vivo OI with a fluorophore-conjugated lead candidate in experimental mice with hOX40-expressing xenografts demonstrated its specificity and functionality as an imaging probe. ConclusionConsidering the need for advanced probes for noninvasive in vivo monitoring of T cell activation dynamics, we propose, that our hOX40-Nbs have a great potential as imaging probes for noninvasive and longitudinal in vivo diagnostics. Quantification of OX40+ T cells in TME or IME will provide crucial insights into the activation state of infiltrating T cells, offering a valuable biomarker for assessing immune responses, predicting treatment efficacy, and guiding personalized immunotherapy strategies in patients with cancer or IMIDs.

immunology↗

bioRxiv2023

Active Suppression of the Nigrostriatal Pathway during Optogenetic Stimulation Revealed by Simultaneous fPET/fMRI

The dopaminergic system is a central component of the brains neurobiological framework, governing motor control, reward responses, and playing an essential role in various brain disorders such as Parkinsons disease and schizophrenia. Within this complex network, the nigrostriatal pathway represents a critical circuit for dopamine transmission from the substantia nigra to the striatum, a connection that is vital to understanding many of the disease-related dysfunctions. However, stand-alone functional magnetic resonance imaging (fMRI) is unable to study the intricate interplay between brain activation and its molecular underpinnings. In our study, the simultaneous use of [18F]FDG functional positron emission tomography (fPET)/BOLD-fMRI provided a new insight that allowed us to demonstrate an active suppression of the nigrostriatal activity during optogenetic stimulation via presynaptic autoinhibition. Our in vivo observation emphasizes that the observed BOLD signal depression during neuronal stimulation does not correlate with neuronal inactivity, but results from an active suppression of neuronal firing as shown by the high [18F]FDG signal increase. This result not only illustrates the potential of simultaneous fPET/fMRI to understand the molecular mechanisms of brain function but also provides a new perspective on how neurotransmitters such as dopamine influence hemodynamic responses in the brain.

neuroscience↗

bioRxiv2023

Two birds with one stone: human SIRPα nanobodies for functional modulation and in vivo imaging of myeloid cells

Signal-regulatory protein (SIRP) expressed by myeloid cells is of particular interest for therapeutic strategies targeting the interaction between SIRP and the "dont eat me" ligand CD47 and as a marker to monitor macrophage infiltration into tumor lesions. To address both approaches, we developed a set of novel human SIRP (hSIRP)-specific nanobodies (Nbs). We identified three high-affinity Nbs targeting the hSIRP/hCD47 interface, thereby enhancing antibody-dependent cellular phagocytosis (ADCP). For non-invasive in vivo imaging, we chose S36 Nb as a non-modulating binder. By quantitative positron emission tomography (PET) in novel hSIRP/hCD47 knock-in (KI) mice, we demonstrated the applicability of 64Cu-hSIRP-S36 Nb to visualize tumor infiltration of myeloid cells. We envision that the hSIRP-Nbs presented in this study have potential as versatile probes, including novel myeloid-specific checkpoint inhibitors for combinatorial treatment approaches and for in vivo stratification and monitoring of individual responses during cancer immunotherapies.

immunology↗

bioRxiv2023

Western diet increases brain metabolism and adaptive immune responses in a mouse model of amyloidosis

Diet-induced body weight gain is a growing health problem worldwide, leading to several serious systemic diseases such as diabetes. Because it is often accompanied by a low-grade metabolic inflammation that alters systemic function, dietary changes may also contribute to the progression of neurodegenerative diseases. Here we demonstrate disrupted glucose and fatty acid metabolism and a disrupted plasma metabolome in a mouse model of Alzheimers disease following a western diet using a multimodal imaging approach and NMR-based metabolomics. We did not detect glial-dependent neuroinflammation, however using flow cytometry we observed T cell recruitment in the brains of western diet-fed mice. Our study highlights the role of the brain-liver-fat-axis and the adaptive immune system in the disruption of brain homeostasis due to a Western diet.

immunology↗

bioRxiv2022

Neurovascular Uncoupling: Multimodal Imaging Delineates the Acute Effects of MDMA

Psychedelic compounds have attracted increasing interest in recent years due to their therapeutic potential for psychiatric disorders. Methylenedioxymethamphetamine (MDMA) is currently being investigated in clinical trials to treat post-traumatic stress disorder. To understand the acute effects of psychedelic drugs in vivo, functional MR imaging (fMRI) has been widely used in recent years. Notably, fMRI studies have shown that MDMA leads to inhibition of brain activity, challenging earlier hypotheses indicating mainly excitatory effects. However, interpretation of hemodynamic changes induced by psychedelics is challenging because of the potent vascular effects associated with this class of substances. Therefore, this study aimed to investigate the acute effects of MDMA using simultaneous positron emission tomography (PET)/fMRI in rats. For this purpose, hemodynamic changes measured by BOLD-fMRI were related to alterations in glucose utilization and serotonin transporter (SERT) occupancy, investigated using [18F]FDG functional PET (fPET) and [11C]DASB PET. We demonstrate that MDMA induces global hemodynamic decreases accompanied by localized metabolic increases. Elevated metabolism was found primarily in limbic projection areas involved in emotion processing. Concurrent BOLD-fMRI decreases, also found in extracerebral areas, indicate that the BOLD-fMRI reductions observed in the brain are of vascular, non-neuronal origin. We further show that higher SERT occupancy strongly correlates with regional BOLD-fMRI reductions. Therefore, increased serotonin levels induced by SERT blockage may cause a neurovascular uncoupling. Correct understanding of the in vivo mechanism of MDMA not only supports ongoing research but also warrants a reassessment of previous studies on neuronal effects of psychedelics relying on neurovascular coupling.

neuroscience↗

bioRxiv2021

Combining CRISPR/Cas9 and brain imaging: from genes to proteins to networks

Receptors, transporters and ion channels are important targets for therapy development in neurological diseases including Alzheime[r]s disease, Parkinso[n]s disease, epilepsy, schizophrenia and major depression. Several receptors and ion channels identified by next generation sequencing may be involved in disease initiation and progression but their mechanistic role in pathogenesis is often poorly understood. Gene editing and in vivo imaging approaches will help to identify the molecular and functional role of these targets and the consequence of their regional dysfunction on whole brain level. Here, we combine CRISPR/Cas9 gene-editing with in vivo positron emission tomography (PET) and functional magnetic resonance imaging (fMRI) to investigate the direct link between genes, molecules, and the brain connectome. The extensive knowledge of the Slc18a2 gene encoding the vesicular monoamine transporter (VMAT2), involved in the storage and release of dopamine, makes it an excellent target for studying the gene networks relationships while structurally preserving neuronal integrity and function. We edited the Slc18a2 in the substantia nigra pars compacta of adult rats and used in vivo molecular imaging besides behavioral, histological, and biochemical assessments to characterize the CRISPR/Cas9-mediated VMAT2 knockdown. Simultaneous PET/fMRI was performed to investigate molecular and functional brain alterations. We found that stage-specific adaptations of brain functional connectivity follow the selective impairment of presynaptic dopamine storage and release. Our study reveals that recruiting different brain networks is an early response to the dopaminergic dysfunction preceding neuronal cell loss. Our combinatorial approach is a novel tool to investigate the impact of specific genes on brain molecular and functional dynamics which will help to develop tailored therapies for normalizing brain function. The method can easily be transferred to higher-order species allowing for a direct comparison of the molecular imaging findings.

neuroscience↗

bioRxiv2021

Single-domain antibodies for targeting, detection and in vivo imaging of human CD4+ cells

The advancement of new immunotherapies necessitates appropriate probes to monitor the presence and distribution of distinct immune cell populations. Considering the key role of CD4+ T cells in regulating immunological processes, we generated novel single-domain antibodies (nanobodies, Nbs) that specifically recognize human CD4. After in depth analysis of their binding properties, recognized epitopes, and effects on T cell proliferation, activation and cytokine release, we selected CD4 Nbs that did not interfere with crucial T cell processes in vitro and converted them into immune tracers for non-invasive molecular imaging. By optical imaging, we demonstrate the ability of a high-affinity CD4-Nb to specifically visualize CD4+ cells in vivo using a xenograft model. Furthermore, time-resolved immune positron emission tomography (immunoPET) of a human CD4 knock-in mouse model showed rapid accumulation of 64Cu-radiolabeled CD4-Nb in CD4+ T cell-rich tissues. We propose that the CD4 Nbs presented here could serve as versatile probes for stratifying patients and monitoring individual immune responses during personalized immunotherapy in both cancer and inflammatory diseases.

immunology↗

bioRxiv2021

Striatal and prefrontal D2R and SERT distributions contrastingly correlate with default-mode connectivity

The molecular substrate of resting-state functional connectivity (rs-FC) remains poorly understood. We aimed to elucidate interactions of dopamine D2 receptor (D2R) and serotonin transporter (SERT) availabilities in main dopaminergic and serotonergic projection areas with the default-mode network (DMN) and two other resting-state networks (RSNs), the salience (SN) and sensorimotor networks (SMN). We performed simultaneous PET/fMRI scans in rats using [11C]raclopride and [11C]DASB to image D2R and SERT distributions, showing for the first time direct relationships between rs-FC and molecular properties of the rodent brain. We found negative associations between CPu D2R availability and all RSNs investigated. Strikingly, medial prefrontal SERT correlated both positively with anterior DMN rs-FC and negatively with rs-FC between the other networks, underlining serotonins intricate role in this region. By further elucidating the link between molecular brain properties and its network-level function, our data support future diagnostic and therapeutic strategies. TeaserSimultaneous PET/fMRI indicates direct associations between monoaminergic neurotransmission and brain functional networks.

neuroscience↗

bioRxiv2018

Causal influences of brain metabolic activity reveal interregional progressions in normal aging

During healthy brain aging, different brain regions show anatomical or functional declines at different rates, and some regions may show compensatory increases in functional activity. However, few studies have explored interregional influences of brain activity during the aging process. We proposed a causality analysis framework combining high dimensionality independent component analysis (ICA), Granger causality, and LASSO (least absolute shrinkage and selection operator) regression on longitudinal brain metabolic activity data measured by Fludeoxyglucose positron emission tomography (FDG-PET). We analyzed FDG-PET images from healthy old subjects, who were scanned for at least five sessions with an averaged intersession interval of about one year. The longitudinal data were concatenated across subjects to form a time series, and the first order autoregressive model was used to measure interregional causality among the independent sources of metabolic activity identified using ICA. Several independent sources with reduced metabolic activity in aging, including the anterior temporal lobe and orbital frontal cortex, demonstrated causal influences over many widespread brain regions. On the other hand, the influenced regions were more distributed, and had smaller age related declines or even relatively increased metabolic activity. The current data demonstrated interregional spreads of aging on metabolic activity at the scale of a year, and have identified key brain regions in the aging process that have strong influences over other regions.

neuroscience↗