PD-L1 positive astrocytes attenuate inflammatory functions of PD-1 positive microglia in models of autoimmune neuroinflammation

Multiple Sclerosis (MS) is a chronic autoimmune inflammatory disorder of the central nervous system (CNS). Current therapies mainly target inflammatory processes during acute stages, but effective treatments for progressive MS are limited. In this context, astrocytes have gained increasing attention as they have the capacity to drive, but also suppress tissue-degeneration. Here we show that astrocytes upregulate the immunomodulatory checkpoint molecule PD-L1 during acute autoimmune CNS inflammation in response to aryl hydrocarbon receptor and interferon signaling. Using CRISPR-Cas9 genetic perturbation in combination with small-molecule and antibody-mediated inhibition of PD-L1 and PD-1 both in vivo and in vitro, we demonstrate that astrocytic PD-L1 and its interaction with microglial PD-1 is required for the attenuation of autoimmune CNS inflammation in acute and progressive stages in a mouse model of MS. Our findings suggest the glial PD-L1/PD-1 axis as a potential therapeutic target for both acute and progressive MS stages.


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The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.Sequencing data have been deposited into the Gene Expression Omnibus (GEO) under the SuperSeries accession number GSE239875.Spatial Transcriptomic data obtained from Hasel et al. is deposited under the Gene Expression Omnibus SuperSeries accession number GSE148612.Clinical data for patient samples can be found in Supplementary Table 1.All supplemental tables with processed data have been submitted with this manuscript.All other data and code that support the findings of this study are available from the corresponding author on reasonable request.
Information on sex for the cohorts used in Figure 1 and 2 has been reported in Table 1.The sex-distrubution in these cohorts reflects the overall prevalence of Multiple Sclerosis.Patient material used for immunohistochemical analysis (Fig. 1d) was obtained from an untreated female MS patient.
Information on sex, age, disease duration, disability (EDSS), and treatment can be found in Table 1.This study was approved by the standing ethical committee (14/18S) at Technical University Munich.Written informed consent was obtained from every patient within the framework of the Biobank resources at the Department of Neurology at Technical University Munich, Germany, which is part of the national competence network Multiple Sclerosis.
N numbers range from n=3 to n=13, with n= individual mouse, based on previously published work with the same stimulation paradigm and readout (PMID: 36266581; PMID: 33888612) No data was excluded from the analysis To ensure replication, all deep sequencing data were repeated in 3-6 mice per group per timepoint.For in vitro and in vivo experiments, at least three independent experiments were performed.
Samples and mice were randomly allocated into biological groups.
Experimenters were blinded to biological group during EAE scoring.Immunohistochemical analyses were performed blinded.For in vitro experiments, no blinding was required as it would not affect of the quantitive results (e.g.RT-qPCR analysis, Flow cytometric analysis, etc.).

Figure 1d :
Figure 1d: All control individuals and those with MS, or their next of kin, had given informed consent for autopsy and use of their brain tissue for research purposes from the MS center (Centre de Resources et de Compétences Sclérose en Plaques Pays de La Loire) of the Nantes University Hospital (ABM PFS13-003 "Collection sclérose en plaques").

Figure 1h /
Figure 1h / Figure 2p: Patients were prespectively recruited in our neuroimmunology outpatient department for diagnostic procedures including CSF sampling.Patients were only included in this study if the diagnosis of multiple sclerosis was confirmed with detection of inflmmatory changes in the CSF including positive oligoclonal bands.CSF was obtained from the Joint Biobank Munich in the framework of the German Biobank node.