The ß1-adrenergic receptor links sympathetic nerves to T cell exhaustion.

CD8+ T cells are essential components of the immune response against viral infections and tumours, and are capable of eliminating infected and cancerous cells. However, when the antigen cannot be cleared, T cells enter a state known as exhaustion1. Although it is clear that chronic antigen contributes to CD8+ T cell exhaustion, less is known about how stress responses in tissues regulate T cell function. Here we show a new link between the stress-associated catecholamines and the progression of T cell exhaustion through the ß1-adrenergic receptor ADRB1. We identify that exhausted CD8+ T cells increase ADRB1 expression and that exposure of ADRB1+ T cells to catecholamines suppresses their cytokine production and proliferation. Exhausted CD8+ T cells cluster around sympathetic nerves in an ADRB1-dependent manner. Ablation of ß1-adrenergic signalling limits the progression of T cells towards the exhausted state in chronic infection and improves effector functions when combined with immune checkpoint blockade (ICB) in melanoma. In a pancreatic cancer model resistant to ICB, ß-blockers and ICB synergize to boost CD8+ T cell responses and induce the development of tissue-resident memory-like T cells. Malignant disease is associated with increased catecholamine levels in patients2,3, and our results establish a connection between the sympathetic stress response, tissue innervation and T cell exhaustion. Here, we uncover a new mechanism by which blocking ß-adrenergic signalling in CD8+ T cells rejuvenates anti-tumour functions.

Primary cell culture of adult zebrafish spinal neurons for electrophysiological studies.

BACKGROUND: Zebrafish (Danio rerio) are growing in popularity as a vertebrate model organism for the study of spinal neurocircuitry and locomotion. While many studies have used the zebrafish model system for electrophysiological analyses in embryonic and larval stages, there is a growing interest in studying spinal circuits and neurons from adult fish. NEW METHOD: To expand upon the existing toolset available to the zebrafish research community, we have developed the first primary cell culture system of adult zebrafish spinal neurons. The intact spinal cord is dissected, and neurons are isolated through enzymatic digestion and mechanical dissociation. Identifiable neurons are viable for electrophysiological analyses after two days in culture. RESULTS: Spinal neurons in culture were confirmed by immunofluorescence labeling and found to exhibit distinct morphologies from other cell types, allowing neurons to be identified based on morphology alone. Neurons were suitable for calcium imaging and whole cell patch clamp recordings, which revealed excitable cells with voltage-gated whole cell currents, including tetrodotoxin-sensitive sodium currents. COMPARISON WITH EXISTING METHODS: This primary cell culture system is the only methodology available to isolate neurons from the adult zebrafish spinal cord. Other methods rely on keeping the spinal cord intact or the utilization of embryonic or larval stage fish. This method provides a robust platform for use in neurophysiological and pharmacological studies. CONCLUSIONS: The novel primary cell culture system described here provides the first in vitro methodology available to isolate and culture neurons from the adult zebrafish spinal cord for use in electrophysiological analyses.