Unveiling EBV-Induced B Cell Migration: The Critical Roles of FAK and IL-10

Epstein-Barr Virus (EBV), a double-stranded DNA virus from the herpesvirus family, was first identified in 1964 by Epstein and Barr in Burkitt lymphoma cells. As the first human tumor virus discovered, EBV is notorious for infecting B cells and contributing to cancers and autoimmune diseases, such as multiple sclerosis. Its ability to persist through latent infection and periodic reactivation allows it to remain in the host long-term, with no effective preventive vaccine or treatment to clear the infection currently available. EBV typically enters the body through the oropharynx, infecting tonsillar B cells, yet infected B cells are found in distant organs like the gut, liver, and brain. This observation prompted a German research team to investigate a key question: How do EBV-infected B cells migrate to these distant tissues? Does the virus alter their migration behavior? This question drove the team to explore how EBV induces abnormal B cell migration.

In Vitro Migration Studies

The research team designed in vitro experiments to simulate B cell migration in a three-dimensional environment. They embedded EBV-infected B cells, resting B cells, CD40L/IL-4-stimulated B cells, and CD40L/IL-4/CXCL12-stimulated B cells (B blasts, as a positive control) in a 3D collagen matrix and observed their movement using time-lapse microscopy. The results were striking: EBV-infected B cells and B blasts exhibited high migration speeds (averaging 8 μm/min) with long, linear trajectories indicative of directed (ballistic) motion, while resting B cells or those stimulated only with CD40L/IL-4 showed minimal movement.

Fig. 1. Time-lapse microscopy of CD40L/IL-4/CXCL12-stimulated B cells, EBV-infected B cells, primary resting B cells, and CD40L/IL-4-stimulated B cells over 15 minutes

Molecular Mechanisms of Migration

Having confirmed the migratory behavior of EBV-infected B cells, the team sought to identify the molecular mechanisms driving this abnormal migration. Previous studies noted that EBV-infected B cells secrete chemokines (e.g., CCL3, CCL4, CCL5) and express chemokine receptors (e.g., CCR1, CCR7). The team hypothesized that EBV might regulate B cell migration through an autocrine chemotaxis loop, particularly via the interaction of CCL4 with its receptor CCR1.

To test this, they conducted Transwell chemotaxis assays, placing CCL4 in the lower chamber to observe whether it attracted EBV-infected B cells. The results showed that CCL4 significantly induced migration of EBV-infected B cells, while inhibiting CCR1 markedly reduced migration. Surprisingly, CCR1 inhibition also significantly decreased B cell proliferation, revealing an unexpected link between migration and proliferation.

Fig. 2. Transwell assay with CCL4 as an attractant, showing EBV-infected B cell migration driven by CCL4 and CCR1

Transendothelial Migration and FAK

The team noted that for B cells to reach distant organs, they must cross endothelial barriers, such as the blood-brain barrier or vascular endothelium, a complex process involving interactions between B cells and endothelial cells. Literature suggests that EBV-infected B cells may induce endothelial cells to express adhesion molecules (e.g., ICAM-1) and disrupt endothelial barrier integrity. The team hypothesized that the CCL4/CCR1 pathway might also contribute, potentially involving downstream signaling molecules like focal adhesion kinase (FAK), which regulates cell movement in chemokine receptor signaling.

Their findings confirmed that EBV-infected B cells, via the CCL4/CCR1 pathway, induced endothelial cells to express ICAM-1, disrupting barrier integrity (e.g., abnormal ZO-1 protein) and promoting transendothelial migration. FAK was identified as a critical regulator in this process. Using the FAK inhibitor Defactinib significantly blocked B cell migration, transendothelial migration, and the growth and survival of EBV-transformed B cells. Additionally, the team discovered that EBV-infected B cells secrete IL-10, which attracts EBV-negative CD52highCD11c+ B cells (with autoimmune characteristics) and enhances their transendothelial migration. Single-cell RNA sequencing and Transwell assays further validated IL-10’s role.

Fig. 3. EBV-infected B cell migration depends on FAK (Defactinib, FAK inhibitor)

In Vivo Validation

To translate these in vitro findings to a more physiological setting, the team used an NSG mouse model to simulate the spread of EBV-infected B cells. They injected EBV-infected B cells (M81 strain, MOI=3) into mice and, after 14 days, began treatment with Defactinib (15 mg/kg, intraperitoneal). Six weeks later, qPCR analysis of spleen EBV BALF5 gene copy numbers and EBER in situ hybridization confirmed the distribution of EBV-infected cells. Defactinib treatment significantly reduced the spread of EBV-infected B cells to the spleen and brain, with markedly lower EBV copy numbers. These findings validated FAK’s critical role in regulating B cell migration in vivo and suggested FAK inhibitors as a potential therapeutic strategy for EBV-related diseases.

Fig. 4. Defactinib treatment protects immunocompromised mice from EBV-infected B cell invasion of the spleen

This study not only elucidated the molecular mechanisms (CCL4/CCR1, FAK, IL-10) behind EBV-induced B cell migration but also highlighted FAK inhibition as a potential therapeutic strategy, offering new directions for treating lymphoma and multiple sclerosis.

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