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Acute Lymph Node Slices Are a Functional Model System to Study Immunity Ex Vivo.

ABSTRACT: The lymph node is a highly organized and dynamic structure that is critical for facilitating the intercellular interactions that constitute adaptive immunity. Most ex vivo studies of the lymph node begin by reducing it to a cell suspension, thus losing the spatial organization, or fixing it, thus losing the ability to make repeated measurements. Live murine lymph node tissue slices offer the potential to retain spatial complexity and dynamic accessibility, but their viability, level of immune activation, and retention of antigen-specific functions have not been validated. Here we systematically characterized live murine lymph node slices as a platform to study immunity. Live lymph node slices maintained the expected spatial organization and cell populations while reflecting the 3D spatial complexity of the organ. Slices collected under optimized conditions were comparable to cell suspensions in terms of both 24-h viability and inflammation. Slices responded to T cell receptor cross-linking with increased surface marker expression and cytokine secretion, in some cases more strongly than matched lymphocyte cultures. Furthermore, slices processed protein antigens, and slices from vaccinated animals responded to ex vivo challenge with antigen-specific cytokine secretion. In summary, lymph node slices provide a versatile platform to investigate immune functions in spatially organized tissue, enabling well-defined stimulation, time-course analysis, and parallel read-outs.

SUBMITTER: Belanger MC

PROVIDER: S-EPMC7887751 | biostudies-literature | 2021 Feb

REPOSITORIES: biostudies-literature

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Acute Lymph Node Slices Are a Functional Model System to Study Immunity Ex Vivo.

Belanger Maura C MC Ball Alexander G AG Catterton Megan A MA Kinman Andrew W L AWL Anbaei Parastoo P Groff Benjamin D BD Melchor Stephanie J SJ Lukens John R JR Ross Ashley E AE Pompano Rebecca R RR

ACS pharmacology & translational science 20210108 1

The lymph node is a highly organized and dynamic structure that is critical for facilitating the intercellular interactions that constitute adaptive immunity. Most ex vivo studies of the lymph node begin by reducing it to a cell suspension, thus losing the spatial organization, or fixing it, thus losing the ability to make repeated measurements. Live murine lymph node tissue slices offer the potential to retain spatial complexity and dynamic accessibility, but their viability, level of immune ac ...[more]

PMID: 33615167

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Project description:Lymph nodes (LNs) are common sites of metastatic invasion in breast cancer, often preceding spread to distant organs and serving as key indicators of clinical disease progression. However, the mechanisms of cancer cell invasion into LNs are not well understood. Existing in vivo models struggle to isolate the specific impacts of the tumor-draining lymph node (TDLN) milieu on cancer cell invasion due to the co-evolving relationship between TDLNs and the upstream tumor. To address these limitations, we used live ex vivo LN tissue slices with intact chemotactic function to model cancer cell spread within a spatially organized microenvironment. After showing that BRPKp110 breast cancer cells were chemoattracted to factors secreted by naïve LN tissue in a 3D migration assay, we demonstrated that ex vivo LN slices could support cancer cell seeding, invasion, and spread. This novel approach revealed dynamic, preferential cancer cell invasion within specific anatomical regions of LNs, particularly the subcapsular sinus (SCS) and cortex, as well as chemokine-rich domains of immobilized CXCL13 and CCL1. While CXCR5 was necessary for a portion of BRPKp110 invasion into naïve LNs, disruption of CXCR5/CXCL13 signaling alone was insufficient to prevent invasion towards CXCL13-rich domains. Finally, we extended this system to pre-metastatic TDLNs, where the ex vivo model predicted a lower invasion of cancer cells. The reduced invasion was not due to diminished chemokine secretion, but it correlated with elevated intranodal IL-21. In summary, this innovative ex vivo model of cancer cell spread in live LN slices provides a platform to investigate cancer invasion within the intricate tissue microenvironment, supporting time-course analysis and parallel read-outs. We anticipate that this system will enable further research into cancer-immune interactions and allow isolation of specific factors that make TDLNs resistant to cancer cell invasion, which are challenging to dissect in vivo.

Project description:BackgroundEpilepsy is a prevalent neurological disorder worldwide. It is characterized by an enduring predisposition to generate seizures and its development is accompanied by alterations in many cellular processes. Organotypic slice cultures represent a multicellular environment with the potential to assess biological mechanisms, and they are used as a starting point for refining molecules for in vivo studies. Here, we investigated organotypic slice cultures as a model of epilepsy.MethodsWe assessed, by electrophysiological recordings, the spontaneous activity of organotypic slices maintained under different culture protocols. Moreover, we evaluated, through molecular-based approaches, neurogenesis, neuronal death, gliosis, expression of proinflammatory cytokines, and activation of NLRP3 inflammasome (nucleotide-binding, leucine-rich repeat, pyrin domain) as biomarkers of neuroinflammation.ResultsWe demonstrated that organotypic slices, maintained under a serum deprivation culture protocol, develop epileptic-like activity. Furthermore, throughout a comparative study with slices that do not depict any epileptiform activity, slices with epileptiform activity were found to display significant differences in terms of inflammation-related features, such as (1) increased neuronal death, with higher incidence in CA1 pyramidal neurons of the hippocampus; (2) activation of astrocytes and microglia, assessed through western blot and immunohistochemistry; (3) upregulation of proinflammatory cytokines, specifically interleukin-1β (IL-1β), interleukin-6, and tumor necrosis factor α, revealed by qPCR; and (4) enhanced expression of NLRP3, assessed by western blot, together with increased NLRP3 activation, showed by IL-1β quantification.ConclusionsThus, organotypic slice cultures gradually deprived of serum mimic the epileptic-like activity, as well as the inflammatory events associated with in vivo epilepsy. This system can be considered a new tool to explore the interplay between neuroinflammation and epilepsy and to screen potential drug candidates, within the inflammatory cascades, to reduce/halt epileptogenesis.

Dataset Information

Acute Lymph Node Slices Are a Functional Model System to Study Immunity Ex Vivo.

Publications

Acute Lymph Node Slices Are a Functional Model System to Study Immunity Ex Vivo.

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