Epstein-Barr virus perpetuates B cell germinal center dynamics and generation of autoimmune-associated phenotypes in vitro

Abstract

Human B cells encompass functionally diverse lineages and phenotypic states that contribute to protective as well as pathogenic responses. Epstein-Barr virus (EBV) provides a unique lens for studying heterogeneous B cell responses, given its adaptation to manipulate intrinsic cell programming. EBV promotes the activation, proliferation, and eventual outgrowth of host B cells as immortalized lymphoblastoid cell lines (LCLs) in vitro, which provide a foundational model of viral latency and lymphomagenesis. Although cellular responses and outcomes of infection can vary significantly within populations, investigations that capture genome-wide perspectives of this variation at single-cell resolution are in nascent stages. We have recently used single-cell approaches to identify EBV-mediated B cell heterogeneity in de novo infection and within LCLs, underscoring the dynamic and complex qualities of latent infection rather than a singular, static infection state. Here, we expand upon these findings with functional characterizations of EBV-induced dynamic phenotypes that mimic B cell immune responses. We found that distinct subpopulations isolated from LCLs could completely reconstitute the full phenotypic spectrum of their parental lines. In conjunction with conserved patterns of cell state diversity identified within scRNA-seq data, these data support a model in which EBV continuously drives recurrent B cell entry, progression through, and egress from the Germinal Center (GC) reaction. This “perpetual GC” also generates tangent cell fate trajectories including terminal plasmablast differentiation, which constitutes a replicative cul-de-sac for EBV from which lytic reactivation provides escape. Furthermore, we found that both established EBV latency and de novo infection support the development of cells with features of atypical memory B cells, which have been broadly associated with autoimmune disorders. Treatment of LCLs with TLR7 agonist or IL-21 was sufficient to generate an increased frequency of IgD-/CD27-/CD23-/CD38+/CD138+ plasmablasts. Separately, de novo EBV infection led to the development of CXCR3+/CD11c+/FCRL4+ B cells within days, providing evidence for possible T cell-independent origins of a recently described EBV-associated neuroinvasive CXCR3+ B cell subset in patients with multiple sclerosis. Collectively, this work reveals unexpected virus-driven complexity across infected cell populations and highlights potential roles of EBV in mediating or priming foundational aspects of virus-associated immune cell dysfunction in disease.

An EBV-associated atypical B cell signature in clinically isolated syndrome is implicated in progression of multiple sclerosis

“…A smaller subset of CD19 + /CD20 + /TBX21 + /ITGAX + cells from patients with CIS (~0.1% of B cells) that exhibited surprisingly broad immune lineage expression in addition to indicators of cellular senescence was absent or significantly underrepresented in long-term non-progressors. While definitive causality cannot be established in the absence of direct viral detection, the data presented here and prior work implicate EBV in a model wherein infection potentiates ABC migration and neuroinvasion by inducing expression of CXCR3 47,50,65 and possibly other chemotactic receptors. The significant enrichment of neuronal genes including cell adhesion molecules in the EBV-associated ABC phenotype suggest an acquired neurotropic capacity that may facilitate CNS-localized viral antigen presentation and inflammatory responses that precipitate development of MS. Because ABCs differentiate into plasmablasts in response to innate and inflammatory stimuli 9 , it is further conceivable that EBV infection might promote low affinity antibody production by ABCs in the CNS.…”

“…Likewise elevated CXCR3 expression, which in T cells promotes migration from lymph nodes to inflammatory sites 100,101 , has been defined as a characteristic of neuroinvasive B cells in several contexts including MS 65,102,103 . Because CXCR3 + B cell frequency in MS is positively correlated with EBV viral load 65 and the virus can induce B cell CXCR3 mRNA and protein expression 33,47 , EBV infection per se may promote ABC trafficking to the central nervous system. Likewise, the observed induction of CD6 expression could conceivably facilitate the niche’s neuroinvasive propensity.…”

“…In addition to its contribution to the development of multiple cancers 34 , EBV is frequently associated with each of the autoimmune and chronic infectious diseases that exhibit clonally expanded ABCs [35][36][37][38][39][40][41][42][43][44][45][46] . We recently reported that EBV can infect existing ABCs de novo 33 and promote their formation in models of latent infection in vitro 47 . However, the manifestation and mechanistic contributions of EBV infection in autoimmune and chronic infectious diseases -including whether they are cell niche-dependent -are incompletely understood.…”

“…Notably, ABCs exhibited variable CXCR3 expression across diseases. This variation may reflect origins from distinct B cell lineages 75 or resting (CXCR3 lo ) versus stimulation-induced activated or differentiated (CXCR3 hi ) phenotypes 47,76,77 . We also confirmed expression of top differentially expressed markers of ABCs previously identified in publicly available datasets (ITGB2, MPP6, NEAT1, PLEK, S100A11, and TNFRSF1B) within CIS cohort and de novo EBV infection samples (Fig.…”

Epstein–Barr virus as a leading cause of multiple sclerosis: mechanisms and implications

“…Numbers of EBV-specific CD8 + T cells were found to be increased in the CSF of patients with MS but possibly not more than in other inflammatory neurological diseases 110,112 . In patients with MS, upregulation of TBX21, CXCR3 and CXCL10 expression through EBV infection of B cells 154,155 as well as the proliferation of CXCR3 + memory B cells following EBV reactivation 156 might allow these memory B cells to gain access to meningeal tertiary lymphoid follicles and the brain parenchyma 138,157 . Blocking the antiviral cytokine IFNγ compromised the antigen-presenting capacity of this subset of B cells 138 .…”