Editor's evaluation: MHC class I and MHC class II reporter mice enable analysis of immune oligodendroglia in mouse models of multiple sclerosis

Abstract

Full text Figures and data Side by side Abstract Editor's evaluation eLife digest Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract Oligodendrocytes and their progenitors upregulate MHC pathways in response to inflammation, but the frequency of this phenotypic change is unknown and the features of these immune oligodendroglia are poorly defined. We generated MHC class I and II transgenic reporter mice to define their dynamics in response to inflammatory demyelination, providing a means to monitor MHC activation in diverse cell types in living mice and define their roles in aging, injury, and disease. Editor's evaluation This study reports an important new resource, MHC class I and MHC class II reporter mice, which provide a means to monitor MHC activation in vivo. The authors use these mice to study inflammatory demyelination in two mouse models of multiple sclerosis. The study provides a compelling demonstration of the new reporter lines as valuable tools for the analysis of inflammation and neurodegeneration. https://doi.org/10.7554/eLife.82938.sa0 Decision letter Reviews on Sciety eLife's review process eLife digest Nerve cells in the brain and spinal cord are surrounded by a layer of insulation called myelin that allows cells to transmit messages to each other more quickly and efficiently. This protective sheath is produced by cells called oligodendrocytes which together with their immature counterparts can also repair damage caused to myelin. In the inflammatory disease multiple sclerosis (MS), this insulation is disrupted and oligodendroglia fail to repair breaks in the myelin sheath, leaving nerves vulnerable to further damage. Recently it was discovered that mature and immature oligodendrocytes (which are collectively known as oligodendroglia) sometimes express proteins normally restricted to the immune system called major histocompatibility complexes (or MHCs for short). Researchers believe that MHC expression may allow oligodendroglia to interact with immune cells, potentially leading to the removal of oligodendroglia by the immune system as well as inflammation that exacerbates damage to nerves and hinders myelin repair. Knowing when oligodendroglia start producing MHCs and where these MHC-expressing cells are located is therefore important for understanding their role in MS. However, it is difficult to identify the location of MHC-expressing oligodendroglia using methods that are currently available. To address this, Harrington, Catenacci et al. created a genetically engineered mouse model in which the MHC-expressing oligodendroglia also generated a red fluorescent protein that could be detected under a microscope. This revealed that only a small number of oligodendroglia in the nervous system had MHCs, but these cells were located in areas of the brain and spinal cord with the highest inflammatory activity. Further microscopy studies in mice that developed MS-like symptoms revealed that MHC production in oligodendroglia increased compared with healthy animals, and that the proportion of oligodendroglia that produced MHC was highest in mice with the most severe symptoms. MHC-expressing oligodendroglia also congregated in the most damaged areas of the brain and spinal cord. These results suggest that MHC expression may contribute to inflammation and impact the function of oligodendroglia that have these molecules. In the future, Harrington et al. hope that their new mouse model will help researchers study the role of MHC expression in different diseases, and in the case of MS, aid the development of new treatments. Introduction Single-cell and single-nucleus RNA sequencing has revealed that some oligodendroglia in both mouse inflammatory models (Falcão et al., 2018; Meijer et al., 2022) and human multiple sclerosis (MS) (Schirmer et al., 2019; Jäkel et al., 2019; Absinta et al., 2021) express transcripts associated with major histocompatibility complex (MHC) antigen presenting and processing pathways. These immune oligodendrocyte precursor cells (iOPCs) and oligodendrocytes (iOLs) have been detected in Alzheimer’s disease (Lau et al., 2020) and viral infection models (Pan et al., 2020; Malone et al., 2008; Phares et al., 2009), and can be induced by exposure to interferon-γ (IFN-γ), suggesting that some oligodendroglia undergo this distinct phenotypic change in response to inflammation. The role of these immune oligodendroglia is unknown (Harrington et al., 2020), but their presence raises the possibility that oligodendroglia may present antigens to T cells, be subject to cytotoxic CD8 T cell-mediated death (Kirby et al., 2019) and perpetuate the immune response through release of cytokines and interactions with CD4 T cells. This oligodendroglial death or inflammation could contribute to impaired remyelination seen in MS and other progressive diseases (Lubetzki et al., 2020; Mahad et al., 2015; Lassmann et al., 2012). Further exploration of the spatial and temporal dynamics of iOPCs/iOLs have been limited by their relative rarity and our inability to identify which cells have transformed in living tissue. To enable detection of which cells upregulate MHC pathways in vivo, we generated two novel MHC I and MHC II reporter mouse lines that express tdTomato when these pathways are activated, and used these lines to define their incidence and transcriptional characteristics in two mouse models of inflammatory demyelination. Results MHC reporter mice are a reliable readout of MHC protein expression MHC class II chaperone invariant chain (Cd74 or Ii) and MHC class I component beta-2-microglobulin (B2m) are required components of the antigen processing/presentation machinery that are expressed by a subset of oligodendroglia in mouse inflammatory models (Falcão et al., 2018) and human MS brain (Schirmer et al., 2019; Jäkel et al., 2019). We chose to target B2m and Cd74 genes for the generation of MHC class I and MHC class II reporter mice, respectively, as both of these transcripts are upregulated in oligodendroglia in response to IFN-γ treatment (Kirby et al., 2019), and both transcripts are present in inflammatory oligodendroglia in mouse inflammatory models (Falcão et al., 2018) and human MS tissue (Schirmer et al., 2019; Jäkel et al., 2019). To facilitate identification of these cells, we used CRISPR/Cas9-mediated gene editing to replace the stop codon of these genes with a P2A-TdTomato-WPRE-pA sequence (Figure 1A-C), generating B2m-tdTomato (B2mtdT) and Cd74-tdTomato (Cd74tdT) reporter mice in which 2A cleaving self-peptide facilitates cleavage of tdTomato from the endogenous protein without disrupting expression of the endogenous genes (Figure 1—figure supplement 1) or antigen presentation (Figure 1—figure supplement 2). Figure 1 with 2 supplements see all Download asset Open asset Major histocompatibility complex (MHC) reporter mice validation. (A) Stop codons of Cd74 and B2m are replaced by P2A-TdTomato-WPRE-pA construct to generate Cd74tdT and B2mtdT reporter mice, respectively. Figure made in https://www.biorender.com/. (B) Genotyping of reporter mice with primers (indicated by arrows in panel A) spanning reporter construct insertion site. (C) Representative histogram of flow cytometry of endogenous tdT expression in peripheral blood CD45+ cells for phenotyping reporter and wild-type mice. (D) Quantification of flow cytometry percentage of endogenous tdT expression in B2mtdT splenic cell populations. n=3 mice. (E) Quantification of flow cytometry percentage of B2m co-expression in tdT-positive CD3+ T cells in B2mtdT spleen with representative histogram of B2m expression with and without B2m antibody. n=3 mice. (F) Quantification of flow cytometry percentage of endogenous tdT expression in Cd74tdT splenic cell populations. n=3 mice. (G) Quantification of flow cytometry percentage of Cd74 co-expression in tdT-positive splenic cell populations in Cd74tdT spleen with representative histogram of tdT-positive CD19+ B cells stained with and without CD74. n=3 mice. (H) Quantification of flow cytometry percentage of I/A-I/E co-expression with tdT-positive splenic cell populations in Cd74tdT spleen with representative histogram of tdT and I-A/I-E expression in monocytes with tdT-intermediate I-A/I-E-negative population outlined. n=3 mice. (I) Immunopanned B2mtdT post-natal pup glial culture with representative images of tdT expression in Olig2+ oligodendroglia with and without interferon-γ (IFN-γ) treatment. Scale bars, 50 µm. (J) Immunopanned Cd74tdT post-natal pup glial culture with representative images of tdT expression in Olig2+ oligodendroglia with and without IFN-γ treatment. Scale bars, 50 µm. (K) Representative images of tdT expression in Olig2+ oligodendroglia, GFAP+ astrocytes, and Iba1+ microglia in Cd74tdT glial culture with IFN-γ treatment. Scale bars, 20 µm. Data represented are means ± s.e.m. Figure 1—source data 1 Generation of Cd74tdT and B2mtdT reporter animals. Cd74-P2A-TdTomato-WPRE-pA and B2m-P2A-TdTomato-WPRE-pA construct sequences with 500 bp homology arms (uppercase sequence) used for generating reporter animals. Primers for genotyping B2mtdT and Cd74tdT reporter animals. https://cdn.elifesciences.org/articles/82938/elife-82938-fig1-data1-v2.xlsx Download elife-82938-fig1-data1-v2.xlsx Figure 1—source data 2 Data from analysis of flow cytometry, sheets are labeled with letter corresponding to data panels in Figure 1. https://cdn.elifesciences.org/articles/82938/elife-82938-fig1-data2-v2.xlsx Download elife-82938-fig1-data2-v2.xlsx Figure 1—source data 3 Representative gating strategy from flow cytometry depicted in Figure 1. https://cdn.elifesciences.org/articles/82938/elife-82938-fig1-data3-v2.pdf Download elife-82938-fig1-data3-v2.pdf Figure 1—source data 4 Original gel image files of PCR genotyping for transgenic reporter founder animals for Figure 1B. https://cdn.elifesciences.org/articles/82938/elife-82938-fig1-data4-v2.zip Download elife-82938-fig1-data4-v2.zip To determine if these transgenes accurately report transcriptional activation of MHC components, we examined splenic immune cells known to express MHC class I and II. In B2mtdT reporter mice, tdT was ubiquitously expressed in all immune cells analyzed in the spleen (Figure 1D) and tdT fluorescent cells were immunoreactive to B2m (Figure 1E). In Cd74tdT reporter mice, tdT expression was highest in professional antigen presenting cells, such as dendritic cells and B cells (Figure 1F) and tdT fluorescent cells were immunoreactive to CD74 (Figure 1G). Independent of MHC class II, Cd74 has diverse roles in cell survival, migration and MIF (macrophage migration inhibitory factor) cytokine signaling (Su et al., 2017; Schröder, 2016). Thus, we determined the relationship between tdT and MHC class II expression using I-A/I-E MHC antibodies. I-A/I-E co-expression with tdT was highest in dendritic cells and B cells (Figure 1H) and monocytes that exhibited intermediate tdT fluorescence were also negative or weakly immunoreactive for I-A/I-E expression (Figure 1H), indicating that the level of tdT expression correlates well with MHC class II receptor expression. To determine whether reporter expression is induced in glial cells, immunopanned reporter expressing glial cells were cultured with and without IFN-γ, as we have previously shown that IFN-γ is sufficient to induce MHC class I and II expression in oligodendroglia in vitro (Kirby et al., 2019). Consistent with this observation, IFN-γ increased tdT expression in Olig2 immunoreactive (+) cells in OPC enriched cultures (Fancy et al., 2011) from both B2mtdT and Cd74tdT (Figure 1I–J). In Cd74tdT glial cultures treated with IFN-γ, tdT expression was also found in Iba1+ microglia and GFAP+ astrocytes (Figure 1K). To characterize the adult central nervous system (CNS) baseline MHC expression, we evaluated the brain, spinal cord, and retinal tissues of adult Cd74tdT and B2mtdT reporter mice. In Cd74tdT mice, tdT expression was most prevalent in the meninges and choroid plexus (Figure 2A–D), but was also observed within some microglia in the ventral brain (Figure 2B and E). tdT expression co-localized with I-A/I-E (Figure 2A–C) and Cd74 (Figure 2C) expression in the meninges and choroid plexus. In the CNS of B2mtdT mice, tdT expression was prominent in endothelial cells and microglia throughout the brain, spinal cord, and retina (Figure 3A–G). In addition, some neurons in the cerebellum, septum, hippocampus, and cortex expressed tdT (Figure 3H–L). Within the corpus callosum and cortex, in addition to microglial and endothelial cell expression, there was diffuse parenchymal expression of tdT (Figure 3A–C), possibly arising from projections of tdT+ neurons. Expression of MHC class I by these neurons may allow them to present antigen and render them more prone to death in inflammatory conditions. Figure 2 Download asset Open asset Cd74tdT reporter adult baseline central nervous system (CNS) tdT expression. (A) Representative images of Cd74tdT endogenous tdT reporter expression in adult spinal cord stained for I-A/I-E. Scale bars, 200 µm. (B) Representative images of Cd74tdT endogenous tdT reporter expression in adult brain stained for I-A/I-E. Scale bars, 1 mm. (C) Confocal images of adult brain choroid plexus endogenous tdT stained with Cd74 and I-A/I-E antibodies. Scale bars, 20 µm. (D) Confocal images of choroid plexus endogenous tdT on CD11c+ dendritic cells. Scale bars, 20 µm. (E) tdT expression on subset of Iba1+ microglia in ventral pallidum (magnification of box in B). Scale bars, 50 µm. Cd74tdT adult CNS reporter expression n=6 adult mice. Figure 3 Download asset Open asset B2mtdT reporter baseline central nervous system (CNS) TdT expression. (A) Representative images of B2mtdT endogenous tdT reporter adult expression in adult brain. Scale bar, 1 mm. (B) Co-localization of tdT in B2mtdT corpus callosum with Iba1+ microglia and CD31+ endothelial cells. Scale bars, 200 µm. (C) Co-localization of tdT with Iba1 in B2mtdT cortex. Scale bar, 200 um. (D) B2mtdT endogenous tdT reporter expression in adult spinal cord. Scale bar, 200 µm. (E) Confocal imaging of corpus callosum, striatum, and spinal cord endogenous tdT expression on CD31+ endothelial cells. Scale bars, 20 µm. (F) Confocal imaging of corpus callosum, striatum, and spinal cord endogenous tdT expression on Iba1+ microglia with tdT+Iba1+ cell bodies indicated with arrows. Scale bars, 20 µm. (G) Flat mount adult B2mtdT retina with TdT expression on CD31+ endothelial cells and Iba1+ microglia. Scale bars, 20 µm. (H) B2mtdT endogenous tdT reporter expression in sagittal brain and coronal brain stained with NeuN (boxes indicate regions of interest confocal imaging in panels I–L). Scale bars, 1 mm. (I) Confocal imaging of endogenous tdT+ parvalbumin PV+ neurons, indicated by arrows, in cerebellar molecular layer. Scale bars, 20 µm. (J) Confocal imaging of septal nucleus with scattered tdT+NeuN+ neurons indicated by arrows. Scale bars, 20 µm. (K) Confocal imaging of hippocampal dentate gyrus tdT+NeuN+ neurons. Scale bars, 20 µm. (L) Confocal imaging of cortex layer II/III tdT+NeuN+ neurons. Scale bars, 20 µm. B2mtdT adult CNS reporter expression n=6 adult mice. MHC reporter expression is induced in EAE To evaluate CNS activation of MHC pathways in distinct cell types under inflammatory conditions, we induced experimental autoimmune encephalitis (EAE) by MOG35-55 peptide immunization. Reporter mice subjected to MOG35-55 EAE were sacrificed with EAE presentation ranging from pre-clinical (score 0), tail and hindlimb weakness (score 1–2.5) to complete hindlimb paralysis (score 3–4). With increasing EAE clinical score, there was a concomitant increase in tdT expression in spinal cord lesion areas in both B2mtdT (Figure 4A) and Cd74tdT animals (Figure 4B). tdT was also present in EAE ventral brain meningeal cell clusters and corpus callosum cell clusters in Cd74tdT and B2mtdT animals (Figure 5A–F). tdT was expressed in both infiltrating myeloid cells and microglia in B2mtdT and Cd74tdT EAE brain (Figure 5G). Figure 4 Download asset Open asset B2mtdT and Cd74tdT reporter expression in MOG35-55 peptide immunized experimental autoimmune encephalitis (EAE) spinal cord. (A) B2mtdT reporter MOG35-55 EAE spinal cord stained with Iba1. tdT expression is present in the vasculature and Iba1+ microglia in pre-clinical score 0 animals and more notable parenchymal expression within EAE lesions indicated by DAPI hypercellularity and Iba1 cell clusters. Scale bars, 200 µm. (B) Cd74tdT reporter MOG35-55 EAE spinal cord stained with Iba1. tdT expression is restricted to the meninges in pre-clinical score 0 mice and with increasing EAE clinical score is prominent within EAE lesions. Scale bars, 200 µm. Figure 5 Download asset Open asset B2mtdT and Cd74tdT reporter expression in MOG35-55 peptide immunized experimental autoimmune encephalitis (EAE) brain. (A) Representative images of Cd74tdT MOG35-55 EAE brain with clinical scores >2 demonstrating parenchymal and meningeal tdT expression. Scale bars, 500 µm. (B) Cd74tdT EAE ventral brain meningeal cluster of tdT-positive cells. Scale bar, 200 µm. (C) Cd74tdT EAE corpus callosum with notable tdT+ infiltrate and tdT parenchymal expression. (D) Representative images of B2mtdT MOG35-55 EAE brain with meningeal tdT expression and corpus callosum tdT expression. Scale bars, 500 µm. (E) B2mtdT EAE ventral brain meningeal cluster of tdT-positive cells. Scale bar, 200 µm. (F) B2mtdT EAE corpus callosum with notable tdT+ infiltrate. (G) Cd74tdT and B2mtdT EAE brain with tdT+ microglia (Iba1+TMEM119+ indicated by arrows) and tdT+ infiltrating myeloid cells (Iba1+TMEM119- indicated by asterisks). Scale bars 20 µm. When analyzed by flow cytometry, infiltrating myeloid cells and microglia exhibited tdT expression in the brain and spinal cord of Cd74tdT mice with EAE (Figure 6A and B) and both the proportion of myeloid cells and microglia that expressed tdT (Figure 6A), and the level of tdT expression by these cells (Figure 6B), were higher in co-stimulatory molecule expressing cells compared to non-co-stimulatory molecule expressing cells. In B2mtdT mouse brain, the percentage of tdT expressing microglia was not significantly different between M1 or M2 microglia, defined by CD86 and CD206 expression, respectively (Jurga et al., 2020; Figure 6C and D); however, M1 microglia had significantly higher tdT fluorescence compared to M2 and M1-2 microglia (Figure 5E). Together, this analysis highlights the ability of these reporter mice to reliably identify endogenous and infiltrating cells that upregulate MHC pathways. Figure 6 Download asset Open asset Co-stimulatory molecule expression in myeloid and microglial cells in MOG35-55 peak experimental autoimmune encephalitis (EAE). (A) Quantification of percentage of tdT expression in co-stimulatory molecule positive (CD40+CD86+) compared to co-stimulatory molecule negative (CD40-CD86-) infiltrating myeloid cells (CD45+CD11b+Clec12a+) and microglia (CD45+CD11b+Clec12a-) in Cd74tdT MOG35-55 EAE by flow cytometry. n=3 mice/group. Unpaired t-test. Data represented are means ± s.e.m. (B) Quantification of tdT mean fluorescence intensity (MFI) in tdT+ co-stimulatory positive and negative infiltrating myeloid cells and microglia in Cd74tdT MOG35-55 EAE by flow cytometry. n=3 mice/group. Unpaired t-test. Data represented are means ± s.e.m. (C) Representative gating strategy for M2 (CD86-CD206+), M1 (CD86+CD206-), and M1-2 microglia (CD86+CD206+). (D) Quantification of percentage of tdT expression in M1, M2, and M1-2 microglia in B2mtdT baseline and EAE brain by flow cytometry. n=3 mice/group. Data represented are means ± s.e.m. (E) Quantification of tdT MFI of M1, M2, and M1-2 microglia in B2mtdT baseline and EAE brain. n=3, mice/group. Unpaired t-test. Data represented are means ± s.e.m. Figure 6—source data 1 Data from analysis of flow cytometry, sheets are labeled with letter corresponding to data panels in Figure 6. https://cdn.elifesciences.org/articles/82938/elife-82938-fig6-data1-v2.xlsx Download elife-82938-fig6-data1-v2.xlsx Figure 6—source data 2 Representative gating strategy from flow cytometry depicted in Figure 6. https://cdn.elifesciences.org/articles/82938/elife-82938-fig6-data2-v2.pdf Download elife-82938-fig6-data2-v2.pdf MHC reporter positive immune oligodendroglia correlate with degree of inflammation To determine whether oligodendroglia express MHC reporters in the setting of inflammation, MOG35-55 immunized brain and spinal cord were analyzed from non-immunized baseline, pre-clinical EAE score 0 (8 days post-immunization [dpi]), and early to peak EAE (8–17 dpi) with a range of EAE clinical scores from 1.5 to 3.5. In both B2mtdT and Cd74tdT MOG35-55 EAE spinal cord, Olig2+ oligodendroglia were found to express tdT within EAE lesions (Figure 7A). To quantify oligodendroglial tdT expression, four spinal cord regions (dorsal horn, lateral white matter, dorsal white matter, and central cord) were quantified for Olig2 and endogenous tdT expression in EAE lumbar spinal cord sections (Figure 7B and C). The number of tdT+Olig2+ oligodendroglia was significantly higher in clinically symptomatic compared to pre-symptomatic score 0 animals (Figure 7D) in both B2mtdT (pre: 1.0 ± 0.2%, n=4; post: 6.3 ± 1.8%, n=9, p=0.0056 unpaired Mann-Whitney t-test) and Cd74tdT spinal cord (pre: 0.02 ± 0.01%, n=3; post: 0.6 ± 0.2%, n=10, p=0.0070 unpaired Mann-Whitney t-test). Figure 7 with 2 supplements see all Download asset Open asset Oligodendroglial major histocompatibility complex (MHC) reporter expression in MOG35-55 experimental autoimmune encephalitis (EAE) spinal cord. (A) Representative confocal imaging of tdT-positive oligodendroglia in MOG35-55 EAE B2mtdT and Cd74tdT spinal cord lesions. Endogenous tdT-positive oligodendroglia indicated by arrows. Scale bars, 20 µm. (B) Representative images of B2mtdT and Cd74tdT MOG35-55 spinal cord whole cord and outlined region of interest (ROI) used for analysis of tdT+ oligodendroglia. Scale bars, 200 µm. (C) Diagram of seven ROIs imaged in lumbar spinal cord sections from baseline and EAE spinal cord. Figure made in https://www.biorender.com/. (D) Quantification of percentage of tdT+Olig2+ oligodendroglia in B2mtdT and Cd74tdT spinal cord. Data points are an individual animal mean across all regions analyzed. n=3–4 mice baseline and score 0, n=9–10 EAE score >0. Unpaired Mann-Whitney t-test. Data represented are means ± s.e.m. (E) Representative confocal imaging of lesion and non-lesion ROIs from the same reporter EAE animal with tdT+Olig2+ oligodendroglia indicated by arrows. Scale bars, 20 µm. (F) Quantification of percentage of tdT+Olig2+ oligodendroglia in lesion compared to non-lesion areas in MOG35-55 EAE spinal cord. Data points are an individual animal mean across ROIs with lesions determined by DAPI hypercellularity compared to ROIs without lesions. n=8–9 mice/reporter, paired Wilcoxon t-test. (G) Linear regression of CD45 density on whole spinal cord section compared to percentage of tdT+Olig2+ oligodendroglia in whole spinal cord section in MOG35-55 EAE spinal cord. Data points are an individual spinal cord sections. n=3–4 sections/mouse, 3 mice/reporter. (H) Linear regression of all region mean percentage of tdT+Olig2+ oligodendroglia compared to all region mean tdT mean fluorescent intensity in MOG35-55 EAE spinal cord. n=13 mice/reporter. Data points are individual animals. (I) Representative confocal imaging of adult B2mtdT spinal cord at baseline with endogenous tdT co-localization with Olig2+CC1+ mature oligodendroglia (arrows). Quantification of percentage of CC1+tdT+ Olig2+ oligodendrocytes in B2mtdT baseline adult spinal cord. n=4 mice. Scale bars, 20 µm. Data represented are means ± s.e.m. (J) Quantification of percentage of PDGFRa+tdT+ oligodendroglia in MOG35-55 EAE spinal cord. Data points are an individual animal mean across all regions. n=3–4 mice baseline and score 0, n=6–8 mice EAE score >0. Unpaired Mann-Whitney t-test. Data represented are means ± s.e.m. Figure 7—source data 1 Immunohistochemistry quantification, sheets are labeled with letter corresponding to data panels in Figure 7. https://cdn.elifesciences.org/articles/82938/elife-82938-fig7-data1-v2.xlsx Download elife-82938-fig7-data1-v2.xlsx tdT+ oligodendroglia were more abundant in EAE lesion areas, determined by DAPI hypercellularity, compared to non-lesion areas in B2mtdT EAE mice (p=0.0156 paired Wilcoxon t-test, n=8) but not Cd74tdT EAE mice (p=0.1289 paired Wilcoxon t-test, n=9) (Figure 7E and F). To quantify the abundance of tdT+ oligodendroglia in relation to the degree of inflammatory infiltrates, CD45 density was quantified on whole spinal cord sections. An increase in CD45 density was significantly correlated with a higher percentage of tdT+ oligodendroglia in B2mtdT EAE sections, but not Cd74tdT EAE sections (Figure 7G), suggesting higher concentrations of MHC class I oligodendroglia in regions with higher inflammatory activity. As an alternative measure of inflammatory activity in individual animals, the mean tdT fluorescent intensity of all spinal cord regions of interest (ROIs) imaged was used as a surrogate of overall inflammatory activity, as tdT is expressed on inflammatory infiltrates in both B2mtdT and Cd74tdT reporter animals. The individual animal mean ROI tdT intensity was positively correlated with the individual animal mean percentage of tdT+ oligodendroglia in both B2mtdT and Cd74tdT reporter mice (Figure 7H), highlighting the close correspondence between activation of MHC pathways in oligodendroglia and level of inflammatory activity. Individual animal EAE clinical score was also positively correlated with the mean percentage of tdT+ oligodendroglia in B2mtdT and Cd74tdT EAE spinal cord (Figure 7—figure supplement 1A). To determine whether tdT+ oligodendroglia were oligodendrocyte progenitors or mature oligodendrocytes, we performed immunostaining for CC1 and PDGFRa, to identify oligodendrocytes and OPCs, respectively. In naive B2mtdT spinal cords, tdT+ oligodendroglia were predominantly mature CC1+ oligodendroglia (88.6 ± 1.7%, n=4) (Figure 7I). As CC1 labels immune cells as well as mature oligodendrocytes, we were only able to analyze OPCs in EAE sections. A minority of spinal cord TdT+Olig2+ oligodendroglia were immunoreactive to PDGFRa (Figure 7J) in B2mtdT naive (6.2 ± 0.2%, n=3), B2mtdT EAE score 0 (11.8 ± 4.0%, n=4), B2mtdT EAE score >0 (12.1 ± 2.8%, n=8), and Cd74tdT EAE score >0 (16.8 ± 4.6%, n=6), indicating that most of these MHC expressing oligodendroglia had advanced beyond the progenitor stage. The level of inflammatory activity in an individual animal based on EAE clinical score (Figure 7—figure supplement 1B), mean tdT ROI intensity (Figure 7—figure supplement 1C), or mean tdT+ oligodendroglia (Figure 7—figure supplement 1D) were not significantly correlated with the percentage of tdT+PDGFRa+ immune OPCs, suggesting that immune OPCs are not depleted in setting of more robust inflammatory activity. To determine if astrocytes demonstrate expression of MHC reporters in the setting of inflammation, EAE spinal cord sections were immunostained for GFAP and Sox9. MHC reporter expression was found in some Sox9+ astrocytes in EAE lesions of both B2mtdT and Cd74tdT animals (Figure 7—figure supplement 2), suggesting that astrocytes also upregulate MHC in the context of inflammation. In addition to spinal cord tissue, we observed tdT+ oligodendroglia in EAE brains (Figure 8A and B and Figure 8—video 1). To quantify tdT+ oligodendroglia in EAE brain regions, four regions (ventral, midline corpus, corpus horn, and subventricular zone) were imaged and quantified for Olig2 expression and endogenous tdT expression (without immunostaining) (Figure 8C and D). The number of tdT+ oligodendroglia across all brain regions analyzed was significantly higher in clinical scoring EAE animals compared to pre-clinical score 0 animals in both B2mtdT (pre: 1.7 ± 0.7%, n=5; post: 6.1 ± 1.2%, n=9, p=0.0180 unpaired Mann-Whitney t-test) and Cd74tdT mice (pre: ND, n=3; post: 0.5 ± 0.2%, n=11, p=0.0165 unpaired Mann-Whitney t-test) animals (Figure 8E). Similar to the spinal cord analysis, the individual animal mean ROI tdT intensity was also positively correlated with the individual animal mean percentage of tdT+ oligodendroglia in the brain of B2mtdT and Cd74tdT EAE mice (Figure 8F). However, the EAE clinical score was correlated with the number of TdT+ oligodendroglia in B2mtdT brain, but not the Cd74tdT EAE brain (Figure 8—figure supplement 1A). Figure 8 with 2 supplements see all Download asset Open asset Oligodendroglial major histocompatibility complex (MHC) reporter expression in the brain of central nervous system (CNS) inflammatory models. (A) Confocal imaging of tdT reporter-positive oligodendroglia in MOG35-55 experimental autoimmune encephalitis (EAE) B2mtdT and Cd74tdT brain. Endogenous tdT reporter-positive oligodendroglia indicated by arrows. Scale bars, 20 µm. (B) Representative images of MOG35-55 EAE B2mtdT and Cd74tdT brain with corpus callosum TdT-positive infiltrate. Scale bars, 500 µm. (C) Representative images of B2mtdT and Cd74tdT MOG35-55 ventral brain used for region of interest (ROI) analysis of tdT+ oligodendroglia. Scale bars, 50 µm. (D) Diagram of seven ROIs imaged in brain sections. Figure made in https://www.biorender.com/. (E) Quantification of percentage of tdT+Olig2+ oligodendroglia in B2mtdT and