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.

Keywords: B cell; Epstein-Barr virus; atypical memory B cells; autoimmunity; chronic infection; germinal center; lymphoblastoid cells; single-cell.

Figure 1

Isolation of EBV⁺ activated and differentiated B cell phenotypes identified from lymphoblastoid cell line scRNA-seq. (A) Top differentially expressed genes between activated and differentiated B cells from representative LCL scRNA-seq data from prior work (111). (B) Flow cytometry gating strategy to assess LCL heterogeneity through proxy surface marker expression. (C) Sorting of three ICAM/CD27 phenotypes from LCLs. ICAM-1^Hi/CD27^Lo = activated B cells; ICAM-1^Lo/CD27^Lo = intermediate states; ICAM-1^Lo/CD27^Hi = differentiated B cells.

Figure 2

EBV⁺ activated and differentiated cell fractions spontaneously recover parental LCL heterogeneity in culture. (A) Growth curves from ICAM-1^Hi/CD27^Lo, ICAM-1^Hi/CD27^Lo, and ICAM-1^Hi/CD27^Lo LCL fractions after sorting. Data from each day are presented as the mean number of cells normalized to initial population size at day 0 (error bars = standard deviation, n = 3 LCLs per fraction). (B) Representative time-resolved staining for ICAM-1 and CD27 in sorted fractions capture recovery of parental line phenotypic heterogeneity. (C) Fraction-resolved quantification of cell growth and phenotype distribution over six days in culture after sorting.

Figure 3

EBV perpetuates a cycle of B cell GC-like entry, engagement, and exit in vitro. (A) Dynamic phenotypes if interest from FACS experiments. (B) Mapping of ICAM-1^Hi/CD27^Lo and ICAM-1^Lo/CD27^Hi phenotypes within integrated LCL scRNA data (n = 3 LCLs). (C) Identification of additional cell states within LCLs. Representative marker gene UMAPs highlight states corresponding to actively cycling cells (red cluster) and pre-GC activated precursor/early MBCs (purple cluster). (D) Annotated clustering and pseudotime trajectory analysis of dynamic LCL states. Pseudotime scores were calculated from graphs initialized in resting MBCs (ICAM-1^Lo/CD27^Hi) and are presented as UMAP representations and cluster-resolved pseudotime score distributions. Cluster ordered pseudotime identifies cyclical state progression in LCLs. (E) Pseudotime-ordered, phenotype-resolved expression of detected EBV latency genes and EBER transcripts in LCLs. (F) Integration of time-resolved FACS findings, scRNA-seq, and pseudotime dynamics support a model of conserved perpetual germinal center (GC) dynamics across EBV-immortalized cells in vitro.

Figure 4

LCL subsets exhibit co-expression of genes associated with distinct GC phenotypes. (A) Gene module scores for mantle zone (MZ), dark zone (DZ), DZ cycling, light zone (LZ), plasmablast (PB), and antibody-secreting cell (ASC) states across tonsils and LCLs. Trajectories depict the path starting at the MZ phenotype and progressing through DZ, DZ cycling, LZ, post GC PB, and Late ASC. Blue trajectories depict the core GC and red trajectories represent exits from this dynamic. [MZ module = CCR6, CD22, CD69, FCRL4, FCRL5, BANK1, MARCH1; DZ module = AICDA, FOXO1, CXCR4, AURKC, IL2RB; DZ cycling module = TCF3, EZH2, CCND3, E2F2, TP53, PLK4, BRCA1; LZ module = NFKB1, NFKB2, CD80, CD83, CD86, BCL2A1, EBI3, CD40, CR2, MIR155HG, ACKR3, MYO1C, MYC; PB module = PRDM1, XBP1, MZB1, TNFRSF17, CD27, CD38; Late ASC = SDC1] (B) Detailed example of a GC-like cell paths in GM12878. Violin plots depicting key gene expression are presented to highlight the core GC dynamic (blue trajectory and box) as well as terminal differentiation to Late ASCs (red trajectory and box). The bifurcation point between ASC and GC re-entry trajectories is associated with cluster-resolved expression of IRF4 and PRDM1, with lower expression of these genes associated with perpetual GC re-entry and higher expression associated with Late ASCs.

Figure 5

Plasma cell differentiation, growth arrest and quiescence, and viral reactivation define tangent fate trajectories arising from core GC dynamics. (A) Model of extra-GC tangent fate trajectories leading to additional phenotypes observed from LCL scRNA-seq data. Proposed triggers associated with each trajectory are annotated in blue. (B) Differentially expressed markers defining tangent phenotypes in LCLs (PB/PC = plasmablasts/plasma cells, quiescent/arrested cells, and lytic reactivation).

Figure 6

An EBV⁺ pre-GC activated state with hallmarks of T-bet⁺ memory B cells develops in a subset of LCLs. (A) AP-eMBC cluster cells within one in-house LCL dataset exhibits an atypical memory B cell (atMBC) phenotype prone to plasma differentiation. (B) Upregulation of genes encoding receptors for IL-21, IFNG, and TLR7 ligands within identified LCL atMBCs coincide with the AP-eMBC phenotype. (C) Model experimental design to stimulate plasma cell differentiation from AP-eMBC/atMBCs within LCLs. (D) Growth analysis of LCLs treated with 2 μg/mL TLR7 agonist R848 (blue), 10 ng/mL IL-21 (orange), or both (green) versus control treatment (0.1% DMSO, red). Data are presented as mean +/- standard deviation cell counts with intra-replicate normalization to Day 0 across 3 biological replicates per condition. Statistically significance of differences (Day 3 IL-21 vs. DMSO, p = 0.04 and Day 3 R848+IL-21 vs. DMSO, p = 0.016) were calculated using Welch’s t test (two-tailed, paired). (E) Representative CD23 (FCER2) expression in LCLs at Day 4 by treatment group. (F) CD27 and IgD staining of LCLs at Day 4 by treatment group to evaluate the frequency of EBV-infected double-negative (DN) B cells (IgD^-/CD27^-). (G) Gating strategy to identify DN B cell-derived plasma cells (IgD^-/CD27^-/CD23^-/CD38⁺/CD138⁺⁺) and quantification by treatment relative to control treated LCLs at Day 4. Statistical significance was evaluated by Welch’s two-tailed t-test.

Figure 7

EBV de novo infection of peripheral B cells induces a CXCR3⁺/CD11c⁺/FCRL4⁺ population that exhibits classic hyperproliferation. (A) Gating for CD19⁺/CXCR3⁺/CD11c⁺/FCRL4⁺ cells within uninfected B cells enriched from PBMCs. (B) Gating as in A) at Day 2 post-EBV infection. (C) Gating for Day 5 post-EBV infection. (D) Gating for Day 8 post-EBV infection. (E) Cell proliferation and distribution of division number over time by gated populations (all CD19⁺, CD19⁺/CXCR3⁺/CD11c⁺, and CD19⁺/CXCR3⁺/CD11c⁺/FCRL4⁺).

Figure 8

Models of EBV-driven germinal center dynamics and possible roles in priming of T-bet⁺ atypical MBC pathogenesis. (A) The Germinal Center (GC) model of EBV infection in vivo. (B) A model of perpetual GC dynamics within EBV-immortalized B cells in vitro. (C) Models of atMBC behaviors associated with pathogenic autoimmunity and potential EBV-induced priming of these responses.