CD8+ T cells recognizing a neuron-restricted antigen injure axons in a model of multiple sclerosis
Benjamin D.S. Clarkson, … , Liz S. Muschler, Charles L. Howe
Benjamin D.S. Clarkson, … , Liz S. Muschler, Charles L. Howe
Published September 7, 2023
Citation Information: J Clin Invest. 2023;133(21):e162788. https://doi.org/10.1172/JCI162788.
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Research Article Immunology Neuroscience

CD8+ T cells recognizing a neuron-restricted antigen injure axons in a model of multiple sclerosis

Abstract

CD8+ T cells outnumber CD4+ cells in multiple sclerosis (MS) lesions associated with disease progression, but the pathogenic role and antigenic targets of these clonally expanded effectors are unknown. Based on evidence that demyelination is necessary but not sufficient for disease progression in MS, we previously hypothesized that CNS-infiltrating CD8+ T cells specific for neuronal antigens directly drive the axonal and neuronal injury that leads to cumulative neurologic disability in patients with MS. We now show that demyelination induced expression of MHC class I on neurons and axons and resulted in presentation of a neuron-specific neoantigen (synapsin promoter–driven chicken ovalbumin) to antigen-specific CD8+ T cells (anti-ovalbumin OT-I TCR-transgenic T cells). These neuroantigen-specific effectors surveilled the CNS in the absence of demyelination but were not retained. However, upon induction of demyelination via cuprizone intoxication, neuroantigen-specific CD8+ T cells proliferated, accumulated in the CNS, and damaged neoantigen-expressing neurons and axons. We further report elevated neuronal expression of MHC class I and β2-microglobulin transcripts and protein in gray matter and white matter tracts in tissue from patients with MS. These findings support a pathogenic role for autoreactive anti-axonal and anti-neuronal CD8+ T cells in MS progression.

Authors

Benjamin D.S. Clarkson, Ethan M. Grund, Miranda M. Standiford, Kanish Mirchia, Maria S. Westphal, Liz S. Muschler, Charles L. Howe

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Figure 1

Antigen processing and presentation genes are upregulated in neurons during CNS demyelination.

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Antigen processing and presentation genes are upregulated in neurons dur...
(A) Schematic of the microfluidic device used to physically isolate pure axonal fields from neuron cell bodies. Representative fluorescence microscopy image of AAV.Syn.GFP-transduced cortical neurons projecting long axons in such a device. For inflammatory stimulation, IFN-γ was added to the distal axon chamber. (B) Axonal fields of mouse cortical neurons were stimulated with 100 ng/mL IFN-γ or vehicle for 72 hours. RNA was isolated from the cell body chamber and processed by microarray analysis of gene transcripts; 296 genes were identified as significantly upregulated (P < 0.05, fold change> 2; genes listed in Supplemental Figure 1). (C) RT-PCR analysis confirmed that multiple antigen processing and presentation genes were retrogradely upregulated in neurons by axonal IFN-γ stimulation (n = 3 samples per condition, B and C). Photomicrographs of axial (D, G, H, I, J) and coronal (E, F, K, L) sections from Syn.Cre × RPL22 mice stained with DAPI (blue; nuclei) and for anti-hemagglutinin (red; HA-tagged ribosomal subunit of Rpl22) in neurons of cortex, hippocampus, and cerebellum. Boxes indicate high-magnification insets (i–vi). Representative of n = 5 mice. Original magnification, ×5 (D–F) and ×10 (digitally magnified insets in G–L). (M) Differentially expressed genes (DEGs) among mRNAs isolated specifically from neurons in Syn.Cre × RPL22 mouse cerebral cortices 18 days after induction of MOG EAE or after 6 weeks on cuprizone diet (CUP), compared with controls. (N) Venn diagram of DEGs upregulated in M. (O) Heatmap showing levels of selected transcripts undergoing active translation in neurons. (P and Q) Relative expression of MHC class I and associated antigen presentation genes in neurons isolated as above (P) or from retina (Q) at 18 days of EAE or after 6 weeks on cuprizone diet (CUP), compared with controls. Each dot represents an individual animal. (R and S) Representative photomicrographs (×40 magnification) of MHC class I expression (red; H2-Db and/or H2-Kb) in cortical neurons and axons (green; AAV.Syn.GFP) counterstained with DAPI (blue; nuclei) in cuprizone (R) and control mice (S). Representative of n > 5 mice per condition. Error bars are the 95% CI. *P < 0.05; **P < 0.01 by multiple unpaired t tests with Welch’s correction and Benjamini-Hochberg correction for multiple comparisons (C) or Kruskal-Wallis 1-way ANOVA with Dunn’s pairwise comparison (P and Q).