Epstein-Barr virus and the immune microenvironment in multiple sclerosis: Insights from high-dimensional brain tissue imaging

Abstract

Epstein-Barr virus (EBV) is strongly implicated in the pathogenesis of multiple sclerosis (MS), yet its exact role in disease progression remains unclear. Using high-dimensional CO-detection by indexing, a technology for spatial imaging, this study examines the cellular microenvironment of MS lesions in secondary progressive MS and primary progressive MS. We analyzed immune, glial, neuronal, and endothelial cell interactions within MS lesions and normal-appearing white matter across two independent cohorts. Our findings show the enrichment of EBV markers, particularly EBNA1 and LMP1, within MS lesions. EBV-positive cells interact closely with reactive astrocytes, microglia, and neurons. Image analysis confirmed the presence of EBV-positive staining within neurons and glial cells, suggesting a direct role for EBV in neuronal and glial involvement in MS. Additionally, we observed altered immune cell interactions, including reduced associations with macrophages and memory T cells, and enhanced interactions with glial cells. Disruptions in blood-brain barrier integrity were also noted in regions of the MS brain. These results highlight EBV's contribution to immune modulation, glial dysfunction, and neuronal damage in MS, particularly in progressive subtypes. The analysis of MS brain tissue suggests potential therapeutic targets, including antivirals and brain penetrant immune modulators, to address EBV's impact on MS progression.

Keywords: Epstein–Barr virus; high-dimensional profiling; molecular mimicry; multiple sclerosis; tissue profiling.

Fig. 1.

EBV-related changes and interactions in MS lesions. (A) Representative images of EBV-positive CD20⁺ cells in MS brain tissue. (Top) CD20+ gating mask (circular red line) confirmed by staining. (Bottom) Representative image of EBNA1+CD20+ B cell expressing BCL-XL antiapoptotic marker interacting with activated microglia and macrophage. Series of images of the same cells (3 panels in red box): (Top) CD20+ (pink arrow), (Middle) EBNA1+ (red arrow), (Bottom) BCL-XL⁺. (B) EBNA1 marker frequency is higher in MS lesions compared to non-MS controls (mean difference = 0.46, P = 0.0086). Log odds ratio analysis indicates significantly increased interactions between EBNA1+ or LMP1+ cells and reactive astrocytes (P = 0.0048 and P = 0.02, respectively; log odds: EBNA1 = 0.48, LMP1 = 0.36) (Panels C and D). Similarly, EBNA1+ and LMP1+ cells exhibit enhanced interactions with neurons (P = 0.01 and P = 0.04, respectively; log odds: EBNA1 = 0.45, LMP1 = 0.46) (Panels E and F). Analysis of interaction odds ratios further reveals that M2 microglia are more frequently associated with EBNA1+ and LMP1+ cells (P = 0.009 and P = 0.015, respectively; log odds: EBNA1 = 0.56, LMP1 = 0.52), while GlialCaM+AQP4+ astrocytes in PPMS lesions also demonstrate an increased association (log odds = 0.48, P = 0.02) (Panels G and H).

Fig. 2.

Euclidian cell-distance and EBNA1+-positive neurons and glial cells in MS lesions. (A) Heatmap of Euclidean distance analysis comparing cell proximity in MS vs. non-MS. Significant findings (depicted by asterisks) EBNA1+>MAP2+Neun+ (P = 0.03), EBNA2+>GFAP+GlialCaM+ (P = 0.044), MAP2+Neun+> Olig2+ (P = 0.00012), IBA1+CD68+>MAP2+Neun+ (P = 0.001). Immunofluorescence shows EBNA1 localization (red arrows) within. (B) Microglia: (Top) EBNA1⁺ microglia (red arrows) interacting with T cells (CD4⁺ purple arrow, CD8⁺ Green arrow) and activated macrophage (orange). (Bottom) EBNA1⁺TMEM119⁺ microglia. (C) Astrocytes: (Top) EBNA1 is found in GFAP+Vimentin+ Astrocytes; both panels show the same area in the MS brain. (Bottom) EBNA1 is found in astrocyte interacting with neuron soma; both panels show the same area in the brain. (D) Neurons: (Top) Representative image of 100 μm field of view depicting EBNA1⁺ and EBNA1⁻ neurons. (Bottom) Left depicts EBNA1⁺ neurons; right depicts EBNA1+ neurons interacting with IBA1⁺ microglia (yellow).

Fig. 3.

Glial–neuronal interactions and gliosis in MS lesions. (A) Representative multiplexed immunofluorescence images showing neuronal loss in MS brain samples, analyzed using PhenoCycler Fusion. (B) Representative multiplexed immunofluorescence images, showing increased GFAP+ astrocytes (blue) and IBA1+ microglia (white) relative to AQP4+ astrocytes (aqua) in MS samples. (C–E) Log2 odds ratio analysis in non-MS vs. MS brain samples. (F and G) Frequency analysis of GFAP+ astrocytes (F) and IBA1+CD163+ microglial cells (G) in SPMS vs. PPMS brain samples.

Fig. 4.

Immune cell enrichment and interactions in MS lesions. (A) Volcano plot showing frequency enrichment of immune cells in MS lesions compared to non-MS controls. (B) Representative multiplexed immunofluorescence images showing immune cell infiltration in MS lesions (Right) compared to non-MS control tissue (Left). Immune cells such as CD4+ T cells, CD8+ T cells, IBA1+ microglia, and CD68+ macrophages are enriched in MS lesions. (Scale bar: 200 µm.) (C–E) Log2 odds ratio analysis showing enhanced interactions between neurons (MAP2) and various immune cells, including CD4+CD8+ double-positive T cells (log odds = 0.47, P = 0.022), CD45RO+ memory T cells (log odds = 0.45, P = 0.025), IBA1+CD68+HLA-DR+ antigen-presenting microglia (log odds = 0.33, P = 0.027), and CD138+ plasma cells (log odds = 0.38, P = 0.034).

Fig. 5.

Immune cell enrichment and interactions in SPMS vs. PPMS lesions. (A and B) Log2 odds ratio analysis showing increased interactions between oligodendrocytes (MBP) and activated microglia (IBA1+CD68+) and macrophages (CD14+CD11b+CD68+) in MS lesions (CD14+CD11b+CD68+; log odds = 0.26, P = 0.05; IBA1+CD68+; log odds = 0.39, P = 0.01). (C) Representative multiplexed immunofluorescence images showing immune cell clustering around perivascular cuffs throughout SPMS lesion areas (Left) compared to PPMS lesions (Right), where immune cell presence is less abundant. Top panels: Lesion areas at low magnification (200 µm). Bottom panels: High magnification (50 µm) of the same regions, focusing on the perivascular areas where immune cells are clustered.

Fig. 6.

Disruption of BBB integrity in MS lesions. (A) Representative multiplexed immunofluorescence images showing changes in BBB markers in MS lesions (Left) and non-MS controls (Right). The images show Claudin-5 (red) and CD31 (yellow) expression in relation to the perivascular area, with increased BAX (aqua, marked by arrows) expression indicating endothelial apoptosis in MS lesions. (Scale bar: 200 µm.) (B) Frequency analysis of CD31+ endothelial cells in MS lesions compared to NAWM from the same samples, with significant differences observed (mean difference = −0.9, P = 0.036). (C–E) Log2 odds ratio analysis showing reduced associations of Claudin-5+ endothelial cells with Vimentin+ pericytes (log odds = −0.48, P = 0.039), Synaptophysin+ synaptic neurons (log odds = −0.49, P = 0.0073), and CD68+ activated macrophages (log odds = −0.39, P = 0.0143) in MS lesions compared to non-MS controls. (F and G) Log2 odds ratio analysis of diminished interactions between Claudin-5+ endothelial cells and EBNA1+ (log odds = −0.5, P = 0.0024) and EBNA2+ (log odds = −0.57, P = 0.00054) cells in MS lesions compared to non-MS controls.