Neuroinflammation is an escalation factor shared by a vast range of central nervous system (CNS) pathologies, from neurodegenerative diseases to neuropsychiatric disorders. CNS immune status emerges by the integration of the responses of resident and not resident cells, leading to alterations in neural circuits functions. To explore spinal cord astrocyte reactivity to inflammatory threats we focused our study on the effects of local inflammation in a controlled micro-environment, the organotypic spinal slices, developed from the spinal cord of mouse embryos. These organ cultures represent a complex in vitro model where sensory-motor cytoarchitecture, synaptic properties and spinal cord resident cells, are retained in a 3D fashion and we recently exploit these cultures to model two diverse immune conditions in the CNS, involving different inflammatory networks and products. Here, we specifically focus on the tuning of calcium signaling in astrocytes by these diverse types of inflammation and we investigate the mechanisms which modulate intracellular calcium release and its spreading among astrocytes in the inflamed environment. Organotypic spinal cord slices are cultured for two or three weeks in vitro (WIV) and exposed for 6 h to a cocktail of cytokines (CKs), composed by tumor necrosis factor alpha (TNF-α), interleukin-1 beta (IL-1 β) and granulocyte macrophage-colony stimulating factor (GM-CSF), or to lipopolysaccharide (LPS). By live calcium imaging of the ventral horn, we document an increase in active astrocytes and in the occurrence of spontaneous calcium oscillations displayed by these cells when exposed to each inflammatory threat. Through several pharmacological treatments, we demonstrate that intracellular calcium sources and the activation of connexin 43 (Cx43) hemichannels have a pivotal role in increasing calcium intercellular communication in both CKs and LPS conditions, while the Cx43 gap junction communication is apparently reduced by the inflammatory treatments.

has not yet been established hindering insight into the posttreatment TCR landscape of MS patients. To address this important knowledge gap, we tracked peripheral T-cell subpopulations (naive and memory CD4+ and CD8+) across 15 RRMS patients before and after 2 years of continuous treatment (NTZ) and a single treatment course (AHSCT) by high-throughput TCRβ sequencing. We found that the two MS treatments left treatment-specific multidimensional traces in patient TCRβ repertoire dynamics with respect to clonal expansion, clonal diversity, and repertoire architecture. Comparing MS TCR sequences with published datasets suggested that the majority of public TCRs belonged to virus-associated sequences. In summary, applying multidimensional computational immunology to a TCRβ dataset of treated MS patients, we show that qualitative changes of TCRβ repertoires encode treatment-specific information that may be relevant for future clinical trials monitoring and personalized MS follow-up, diagnosis, and treatment regimens.

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Background: T cells play a key role in the pathogenesis of multiple sclerosis (MS), a chronic, inflammatory, demyelinating disease of the central nervous system (CNS). Although several studies recently investigated the T-cell receptor (TCR) repertoire in cerebrospinal fluid (CSF) of MS patients by high-throughput sequencing (HTS), a deep analysis on repertoire similarities and differences among compartments is still missing. Methods: We performed comprehensive bioinformatics on high-dimensional TCR Vb sequencing data from published and unpublished MS and healthy donors (HD) studies. We evaluated repertoire polarization, clone distribution, shared CDR3 amino acid sequences (CDR3s-a.a.) across repertoires, clone overlap with public databases, and TCR similarity architecture. Findings: CSF repertoires showed a significantly higher public clones percentage and sequence similarity compared to peripheral blood (PB). On the other hand, we failed to reject the null hypothesis that the repertoire polarization is the same between CSF and PB. One Primary-Progressive MS (PPMS) CSF repertoire differed from the others in terms of TCR similarity architecture. Cluster analysis splits MS from HD. Interpretation: In MS patients, the presence of a physiological barrier, the blood-brain barrier, does not impact clone prevalence and distribution, but impacts public clones, indicating CSF as a more private site. We reported a high Vb sequence similarity in the CSF-TCR architecture in one PPMS. If confirmed it may be an interesting insight into MS progressive inflammatory mechanisms. The clustering of MS repertoires from HD suggests that disease shapes the TCR Vb clonal profile. Funding: This study was partly financially supported by the Italian Multiple Sclerosis Foundation (FISM), that contributed to Ballerini-DB data collection (grant #2015 R02).