Epstein-Barr Virus-Induced Upregulation of GPR183: A Potential Upstream Mechanism in IgG4-Related Ophthalmic Disease

Abstract

Purpose

Immunoglobulin G4-related ophthalmic disease (IgG4-ROD) is the most common subtype of IgG4-related disease (IgG4-RD), yet its pathogenic mechanisms remain poorly understood. Ectopic germinal centers are a hallmark histopathological feature of IgG4-ROD, but the molecular drivers underlying their formation remain unclear. This study aimed to identify potential upstream triggers of IgG4-ROD and novel therapeutic targets.

Methods

Fourteen patients with IgG4-ROD and 13 controls (9 with lacrimal gland prolapse and 4 with chronic dacryoadenitis) were enrolled. Lacrimal gland specimens were analyzed using bulk RNA sequencing (RNA-seq), multiplex immunofluorescence, flow cytometry, and quantitative real-time PCR (qPCR). Public bulk and single-cell RNA-seq datasets were further analyzed.

Results

GPR183 was the only gene consistently upregulated across lacrimal gland, salivary gland, peripheral blood, and retroperitoneal fibrosis tissues from patients with IgG4-RD. GPR183 was broadly expressed across multiple immune cell populations in lacrimal glands. Multiplex immunofluorescence demonstrated prominent GPR183 expression within ectopic germinal centers, particularly in B-lymphocyte-rich regions. The qPCR revealed a significantly higher Epstein-Barr virus (EBV) positivity rate in IgG4-ROD lacrimal glands than in controls (85.7% vs. 15.4%, P < 0.001). EBV DNA copy number positively correlated with serum IgG4 levels (adjusted R² = 0.79, P < 0.01). EBV markers EBNA1 and LMP1 were co-expressed with GPR183. Single-cell RNA-seq showed that early EBV infection induced GPR183 expression in T and B lymphocytes and was associated with B-lymphocyte differentiation.

Conclusions

Our findings suggest that EBV-induced GPR183 upregulation potentially promotes ectopic germinal center formation and contributes to the pathogenesis of IgG4-ROD.

Immunoglobulin G4-related disease (IgG4-RD) is a systemic immune-mediated disorder that is believed but not confirmed to have an autoimmune origin.¹ IgG4-RD was first recognized as a distinct clinical entity in 2003.² IgG4-RD is considered a rare disease, with an estimated prevalence of 0.28 to 1.08 per 100,000 people.³ IgG4-RD is characterized by fibroinflammatory infiltrates that can affect essentially any organ system.⁴

Immunoglobulin G4-related ophthalmic disease (IgG4-ROD) is a distinct condition within the spectrum of IgG4-RD. Epidemiological studies indicate that IgG4-ROD develops in 54.7% to 58.8% of patients with IgG4-RD.^5,6 IgG4-ROD is characterized by tumor-like enlargement with IgG4-positive plasma cell infiltration of various ophthalmic tissues, most frequently the lacrimal glands.⁷ Lacrimal gland involvement typically manifests as lateral upper eyelid swelling, proptosis, S-shaped ptosis, and dry eye disease. Involvement of the extraocular muscles can result in diplopia. Most seriously, optic nerve involvement may lead to irreversible vision loss.⁸ Although accumulating evidence indicates that dysregulated immune activation plays a pivotal role in the pathogenesis of IgG4-ROD, the underlying mechanisms remain incompletely understood. Accordingly, the first-line treatment for IgG4-ROD is immunosuppressive therapy, most commonly glucocorticoids. However, up to 70% of patients experience disease relapse after the initial course of oral steroids and ultimately require long-term immunosuppressive therapy.⁹

Ectopic germinal centers are among the most characteristic histopathological features of IgG4-ROD.⁷ It is now recognized that aberrant immune responses occurring within these ectopic germinal centers play a critical pathogenic role in IgG4-ROD.¹⁰ In particular, follicular helper T (Tfh) cells orchestrate B lymphocytes by secreting cytokines such as IL-4 and IL-21 and by delivering co-stimulatory signals including ICOS and CD40L, thus promoting the proliferation and differentiation of B lymphocytes.¹¹ In response to Tfh signals, B lymphocytes undergo somatic hypermutation and class-switch recombination, ultimately differentiating into mature IgG4-positive plasma cells. Despite these insights, the mechanisms driving the formation of these ectopic germinal centers remain elusive, representing a major gap in our understanding of IgG4-ROD pathogenesis.

This study integrates bulk RNA sequencing (bulk RNA-seq), multiplex immunofluorescence, flow cytometry, quantitative real-time PCR (qPCR), and single-cell RNA-seq (scRNA-seq) to identify potential upstream triggers of IgG4-ROD and novel therapeutic targets.

Methods

Subjects

This study enrolled 14 patients with IgG4-ROD, together with 9 patients with lacrimal gland prolapse and 4 patients with chronic dacryoadenitis as controls, all of whom presented to the Department of Ophthalmology at Peking University Third Hospital from 2024 to 2026. The diagnosis of IgG4-ROD was established according to the 2023 revised diagnostic criteria.⁷ None of the enrolled patients had received glucocorticoids, immunosuppressive agents, or other disease-specific treatments prior to surgery. The demographic and laboratory features of the patients and controls are summarized in the Table.

Table.

Clinical and Laboratory Features of Immunoglobulin G4-Related Ophthalmic Disease (IgG4-ROD) Patients and Controls

	IgG4-ROD	Lacrimal Gland Prolapse	Chronic Dacryoadenitis
Number	14	9	4
Sex M: F	3: 11	0: 9	1: 3
Age, y	55.2 ± 11.5 (39–74)	34.3 ± 8.7 (22–45)	43.3 ± 4.6 (39–49)
Disease duration, y	1.2 ± 1.3 (0.25–5.0)	3.3 ± 3.2 (0.16–10.0)	1.2 ± 0.6 (0.75–2.0)
IgG4, mg/dL	1189.6 ± 1341.5 (165.0–4880.0)	Not measured	Not measured

Age, disease duration, and IgG4 are shown in mean ± standard deviation (range).

Lacrimal gland specimens were obtained from histopathological biopsies of the patients and controls. All samples were frozen within 30 minutes of collection and stored at –80°C until analysis. This study was approved by the Ethics Committee of Peking University Third Hospital (M2024661) and conducted in accordance with the tenets of the Declaration of Helsinki. The study was registered at ClinicalTrials.gov (NCT06655831). Written informed consent was obtained from all participants. All the raw RNA-seq data used in this study have been deposited in the NCBI Sequence Read Archive (SRA) under BioProject accession number PRJNA1367663 (https://www.ncbi.nlm.nih.gov/sra/PRJNA1367663), and are available under a CC-BY 4.0 license to anyone who wishes to access the data.

Bulk RNA-Seq Analysis for Differentially Expressed Genes and Pathway Analysis

Bulk RNA-seq was performed using lacrimal gland biopsy specimens from five patients with IgG4-ROD and three with lacrimal gland prolapse. Total RNA was extracted using TRIzol reagent, and RNA quality was assessed using the Bioanalyzer 2100 system (Agilent Technologies, Santa Clara, CA, USA). Sequencing libraries were prepared according to standard protocols and sequenced on an Illumina platform to generate 150-bp paired-end reads. Raw sequencing reads were processed using fastp to remove adapter sequences and low-quality reads. Clean reads were aligned to the human reference genome (GRCh38, Ensembl release 110) using HISAT2 version 2.0.5, and gene-level read counts were generated using featureCounts version 1.5.0-p3. Differential expression analysis was performed using DESeq2 version 1.20.0. P values were adjusted using the Benjamini-Hochberg method, and genes with an adjusted P value ≤ 0.05 and an absolute log2 fold change ≥1 were considered differentially expressed. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were conducted using the clusterProfiler package, and pathways with an adjusted P value ≤ 0.05 were considered significantly enriched.

To identify genes that play key pathogenic roles in IgG4-ROD, we performed an integrative analysis of public RNA-seq datasets from different affected sites of IgG4-RD with various controls. These datasets included: bulk RNA-seq of lacrimal glands from patients with IgG4-ROD compared with orbital cavernous hemangioma tissues (GSE76497),¹² bulk RNA-seq of labial salivary glands from patients with IgG4-RD compared with Sjögren's syndrome and healthy controls (GSE40568),¹³ bulk RNA-seq of peripheral blood from patients with IgG4-RD compared with healthy controls (GSE66465),¹⁴ and scRNA-seq of retroperitoneal fibrosis from patients with IgG4-RD compared with benign retroperitoneal tumors (GSE231920).¹⁵ For bulk RNA-seq data, differentially expressed gene (DEG) analysis was performed following the methods described in the respective original publications.^12–14 For scRNA-seq data, pseudobulk profiles were generated using the AggregateExpression function in the Seurat software package, and DEG analysis was conducted using the RankProd method. Genes consistently and significantly upregulated across all affected IgG4-RD sites were considered potential triggers of IgG4-ROD pathogenesis.

Flow Cytometry Analysis of Immune Cells’ Infiltration in the Lacrimal Gland Tissues

Flow cytometry was performed on lacrimal gland tissues obtained from one patient with IgG4-ROD and one lacrimal gland prolapse control, following a previously established protocol from our laboratory.¹⁶ Briefly, lacrimal gland tissues were mechanically minced and enzymatically dissociated in a GentleMACS Dissociator (Miltenyi Biotec, Germany). Single-cell suspensions were then prepared, and immune cells were enriched by Percoll density-gradient centrifugation. The resulting cells were stained with fluorochrome-conjugated antibodies for identification of immune cell subsets. Zombie NIR Fixable Viability Kit (BioLegend, San Diego, CA, USA) was used to exclude dead cells. BUV805-conjugated anti-human CD45 antibody (BD Biosciences Cat# 612892, RRID: AB_2870179) was used to identify total immune cells. PE-Fire 640-conjugated anti-human CD3 (BioLegend Cat# 344859, RRID: AB_2860896) and BUV737-conjugated anti-human CD4 antibodies (BD Biosciences Cat# 612749, RRID: AB_2870080) were used to identify CD4+ T lymphocytes. RB780-conjugated anti-human CD19 antibody (BD Biosciences Cat# 755602, RRID: AB_3687943) was used to identify B lymphocytes. BV570-conjugated anti-human HLA-DR (BD BioLegend Cat# 307637, RRID: AB_10895753) and BB515-conjugated anti-human CD11c antibodies (BD Biosciences Cat# 564491, RRID: AB_2744273) were used to identify conventional dendritic cells (cDCs), while AF594-conjugated anti-human CD14 (BioLegend Cat# 325630, RRID: AB_2563225) antibody was used to identify monocytes/macrophages. An APC-conjugated anti-human GPR183 antibody (BioLegend Cat# 368907, RRID: AB_2632940) was used to detect GPR183+ subsets within the immune cell populations. Flow cytometry acquisition was performed on a FACSCanto Plus flow cytometer (BD Biosciences), and the data were analyzed using FlowJo software (version 10.8.1; Tree Star Inc., Ashland, OR, USA).

Multiplex Opal Immunofluorescence Staining

Multiplex immunofluorescence was performed on 5-µm formalin-fixed paraffin-embedded (FFPE) human lacrimal gland tissue sections using the Opal Multiplex IHC system (Akoya Biosciences, Marlborough, MA, USA) according to the manufacturer's instructions. The following primary antibodies were used for multiplex staining: CD3 (Abcam Cat# ab16669, RRID: AB_443425; 1:200), CD20 (Abcam Cat# ab64088, RRID: AB_1139386; 1:100), PD-1 (Abcam Cat# ab237728, RRID: AB_3073606; 1:200), CD10 (Abcam Cat# ab255609; 1:200), CD21 (Abcam Cat# ab315160, RRID: AB_3697666; 1:50), GPR183 (Invitrogen Cat# PA5-100950, RRID: AB_2850437; 1:200), EBNA1 (Abcam Cat# ab316860, RRID: AB_3661848; 1:200), and LMP1 (Abcam Cat# ab78113, RRID: AB_1566182; 1:100). Images were acquired using the PhenoImager HT system (Akoya Biosciences) and visualized with Phenochart 2.0.0 software (Akoya Biosciences). Automated cell detection and phenotypic classification were performed using QuPath version 0.5.1.¹⁷

qPCR Detection of Viruses in the Lacrimal Gland Tissues

The qPCR was carried out using biopsy specimens from all the 14 patients with IgG4-ROD and the 13 controls. Lacrimal gland tissues were processed using an automated nucleic acid extraction platform (Thermo Fisher Scientific, Plainville, MA, USA). The presence of six viruses commonly associated with ocular infections, Epstein-Barr virus (EBV), cytomegalovirus (CMV), adenovirus (ADV), varicella-zoster virus (VZV), and herpes simplex virus (HSV) types I and II, was evaluated using nucleic acid detection qPCR kits (Liferiver, China). All assays were conducted by experienced clinical laboratory personnel using instruments routinely used for clinical diagnostic testing. According to the manufacturer's specifications, the assay has a lower limit of detection of 1 × 10³ copies/mL, and samples with a viral load of ≥1 × 10³ copies/mL were classified as positive. The assay has been validated for analytical specificity, with no reported cross-reactivity with hepatitis B virus, hepatitis C virus, CMV, human herpesvirus 6, or human T-cell leukemia virus. In addition, its diagnostic accuracy and clinical utility have been well established through extensive routine clinical use.

scRNA-Seq Analysis of EBV Infection in Lymphocytes

The scRNA-seq data from the GSE189141 dataset were analyzed to investigate the effects of EBV infection on human lymphocytes. The dataset contains B lymphocytes-enriched peripheral blood mononuclear cells collected before infection (day 0) and at days 2, 5, and 8 after in vitro EBV infection, with simultaneous detection of both human and EBV transcripts. Data processing and analysis were performed using Seurat version 5.4.0 in R software. After quality control and normalization, highly variable genes were identified and used for principal component analysis (PCA). Cells were clustered using a graph-based approach and visualized using uniform manifold approximation and projection (UMAP). Cell-type annotation was assigned according to the classification strategy described by SoRelle et al.¹⁸ To investigate transcriptional changes during EBV-driven B-lymphocyte differentiation, pseudotime analysis was performed using Monocle3 version 1.4.27. Only B lymphocytes were included in trajectory reconstruction, whereas T lymphocytes and monocytes were excluded. Gene expression dynamics along pseudotime were visualized to characterize temporal changes associated with EBV infection. Figures were generated using ggplot2 version 4.0.3 and related R packages.

Statistical Analysis

The qPCR data were analyzed using SPSS statistical software version 26. The statistical significance of differences in EBV positivity rates was assessed with the chi-square test. The correlation between EBV DNA copy numbers in lacrimal gland tissues of patients with IgG4-ROD and serum IgG4 concentrations was examined using Pearson's correlation coefficient. A 2-tailed P value < 0.05 was considered statistically significant.

Results

GPR183 Was Consistently Upregulated Across Multiple IgG4-RD Lesions

Bulk RNA-seq analysis identified 3722 upregulated and 2644 downregulated DEGs in IgG4-ROD compared with lacrimal gland prolapse controls (Fig. 1A). Because IgG4-ROD is a subtype of IgG4-RD, we hypothesized that genes playing a key pathogenic role in IgG4-ROD should also be differentially expressed across other affected sites of IgG4-RD. In this study, we focused on the upregulated genes. By intersecting the upregulated DEGs from our bulk RNA-seq data with those derived from public datasets of IgG4-RD involving the lacrimal gland, labial salivary glands, peripheral blood, and retroperitoneal fibrosis, we found that GPR183 was the only gene consistently and significantly upregulated across all these lesions (Fig. 1B), with log2 fold changes ranging from 1.05 to 3.81.

Figure 1.

Bulk RNA - s eq analysis of differentially expressed genes (DEGs) in lacrimal gland tissues from patients with immunoglobulin G4-related ophthalmic disease (IgG4-ROD) and controls. (A) Bulk RNA-seq identified 3722 upregulated and 2644 downregulated DEGs in patients with IgG4-ROD (n = 5) compared with lacrimal gland prolapse controls (n = 3). (B) GPR183 was the only gene consistently and significantly upregulated across multiple immunoglobulin G4-related disease (IgG4-RD) affected tissues, including the lacrimal gland, labial salivary glands, peripheral blood, and retroperitoneal fibrosis.

Multiple Immune Cell Populations Contributed to GPR183 Expression in the Lacrimal Glands of Patients With IgG4-ROD

GPR183 can be expressed by multiple immune cell populations. To identify the cellular sources of GPR183 in the lacrimal glands of patients with IgG4-ROD, we performed flow cytometry on infiltrating immune cells isolated from the lacrimal gland of one patient with IgG4-ROD and one lacrimal gland prolapse control. Marked infiltration of both T and B lymphocytes was observed in the IgG4-ROD lacrimal gland. Among these cells, GPR183+ subsets accounted for 64.6% of CD4+ T lymphocytes and 72.4% of B lymphocytes (Fig. 2A). In contrast, infiltrating immune cells in the control lacrimal gland consisted predominantly of T lymphocytes, which represented 97.1% of total immune cells, whereas B lymphocytes were scarce. The proportions of GPR183+ subsets among CD4+ T lymphocytes and B lymphocytes in the control sample were only 5.48% and 18.4%, respectively (Fig. 2B). In addition, GPR183+ subsets were detected among cDCs and monocyte/macrophage populations in the IgG4-ROD lacrimal gland, which were absent in the controls.

Figure 2.

GPR183 was broadly expressed across multiple immune cell populations in the lacrimal gland of patients with immunoglobulin G4-related ophthalmic disease (IgG4-ROD). (A) Representative flow cytometric analysis of infiltrating immune cells in lacrimal gland tissues from patients with IgG4-ROD. (B) Representative flow cytometric analysis of infiltrating immune cells in lacrimal gland tissues from lacrimal gland prolapse control.

GPR183 Was Localized Within Ectopic Germinal Centers in Lacrimal Glands of Patients With IgG4-ROD

To investigate the potential role of GPR183 in IgG4-ROD, we performed multiplex immunofluorescence staining to determine its spatial distribution in lacrimal glands from patients with IgG4-ROD. CD20 and CD10 were used as markers of germinal center B cells (GC B), CD3 and PD-1 as markers of Tfh, and CD21 as a marker of follicular dendritic cells (FDCs). Well-defined ectopic germinal centers composed of GC B, Tfh, and FDC were identified in the lacrimal glands of patients with IgG4-ROD. Notably, GPR183 exhibited a strong signal within these ectopic germinal centers (Fig. 3A). Based on quantitative fluorescence intensity for CD3 and CD20, cells were classified as T lymphocytes, B lymphocytes, or unclassified. Notably, regions with strong GPR183 expression largely overlapped with areas enriched for B lymphocytes (Fig. 3B).

Figure 3.

GPR183 was prominently localized within ectopic germinal centers in the lacrimal glands of patients with immunoglobulin G4-related ophthalmic disease (IgG4-ROD), particularly in B-lymphocyte-rich regions. (A) Multiplex immunofluorescence staining of lacrimal gland tissue. Cyan represents CD20 and white represents CD10, both marking germinal center B cells (GC B cells). Green represents CD3 and red represents PD-1, both marking T follicular helper (Tfh) cells. Yellow represents CD21, a marker of follicular dendritic cells (FDCs). Orange indicates GPR183, and blue represents DAPI. Well-defined ectopic germinal centers composed of GC B cells, Tfh cells, and FDCs were observed, with strong GPR183 expression localized within these structures. (B) In the upper panel, the regions enclosed by the red and yellow outlines indicate areas of strong GPR183 expression. In the lower panel, T and B lymphocytes were identified based on CD3 and CD20 signals, respectively. The GPR183-strong region was found to largely overlap with the B-lymphocyte-rich regions.

EBV Positivity Rate Was Significantly Elevated in Lacrimal Glands of Patients With IgG4-ROD

As described above, we identified GPR183 as a potential key pathogenic gene in IgG4-ROD; however, the cause of its upregulation in lacrimal gland tissues of patients with IgG4-ROD remains unclear. To address this, we performed GO and KEGG enrichment analyses on the upregulated DEGs from patients with IgG4-ROD to explore potential pathways involved. GO enrichment revealed significant enrichment of 52 pathways related to “immune system processes,” “immune responses,” and “G protein-coupled receptor activity,” etc. (Fig. 4A). KEGG enrichment identified 96 significantly enriched pathways, including “hematopoietic cell lineage,” “cytokine-cytokine receptor interaction,” and “primary immunodeficiency,” etc. (Fig. 4B). Notably, KEGG enrichment revealed significant enrichment of virus infection-related pathways, such as “EBV infection” (Fig. 4C). As suggested by its alternative name, EBV-induced G-protein coupled receptor 2 (EBI2), GPR183 was first identified in 1993 as an EBV-induced receptor in Burkitt lymphoma cell lines.¹⁹ Based on these findings, we hypothesize that the elevated expression of GPR183 in lacrimal gland tissues of patients with IgG4-ROD may be attributable to EBV infection.

Figure 4.

Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses of upregulated differentially expressed genes (DEGs) in lacrimal glands from patients with immunoglobulin G4-related ophthalmic disease (IgG4-ROD). (A) The top 15 GO terms ranked by the smallest adjusted P values. (B) The top 15 KEGG pathways ranked by the smallest adjusted P values. (C) KEGG enrichment map of the Epstein-Barr virus (EBV) infection pathway. The color scale indicates the log2 fold enrichment of the corresponding genes in the lacrimal glands of patients with IgG4-ROD compared with controls.

To test this hypothesis, we conducted qPCR analysis of lacrimal gland tissues from 14 patients with IgG4-ROD and 13 controls, focusing on EBV while also screening for 5 other common ocular pathogens (CMV, ADV, VZV, and HSV types I and II). EBV positivity was observed in 85.7% of patients with IgG4-ROD, which was significantly higher than that in the control group (15.4%, P < 0.001; Fig. 5A). All other tested viruses were negative in both groups. EBV DNA copy number in lacrimal gland tissues showed a significant positive correlation with serum IgG4 concentration in patients with IgG4-ROD (R² = 0.85, P < 0.001). Importantly, this association remained significant after adjustment for age, sex, and disease duration, indicating that the relationship between EBV burden and serum IgG4 levels was independent of these potential confounding factors (adjusted R² = 0.79, P < 0.01; Fig. 5B).

Figure 5.

Real-time PCR (qPCR) detection of Epstein-Barr virus (EBV) in the lacrimal gland tissues from patients with immunoglobulin G4-related ophthalmic disease (IgG4-ROD) and controls. (A) EBV positivity rate was significantly elevated in the lacrimal glands of patients with IgG4-ROD (n = 14) than in controls (n = 13, 85.7% vs. 15.4%, P < 0.001). (B) EBV DNA copy number in the lacrimal glands of patients with IgG4-ROD (n = 14) positively correlated with serum IgG4 concentration (adjusted R² = 0.79, P < 0.01).

GPR183 Was Co-expressed With EBV Infection Markers in Lacrimal Glands of Patients With IgG4-ROD

To investigate the correlation between upregulated GPR183 expression and EBV infection in IgG4-ROD, we performed multiplex immunofluorescence staining. EBNA1 and LMP1 were used as markers for EBV infection. A substantial number of EBNA1+LMP1+ double-positive cells were detected in the lacrimal glands of patients with IgG4-ROD, whereas little or no EBNA1 or LMP1 signal was observed in lacrimal gland prolapse controls, consistent with the qPCR findings. Furthermore, co-expression of GPR183 with both EBNA1 and LMP1 was observed (Fig. 6).

Figure 6.

Co-expression of GPR183 and Epstein-Barr virus (EBV) markers in lacrimal glands of patients with immunoglobulin G4-related ophthalmic disease (IgG4-ROD). EBNA1 is shown in yellow and LMP1 in red, both serving as markers of EBV infection. No detectable EBV marker expression was observed in lacrimal gland tissues from lacrimal gland prolapse control. In contrast, numerous EBNA1+ LMP1+ double-positive cells, indicative of EBV infection, were identified in lacrimal gland tissues from patients with IgG4-ROD. Moreover, co-expression of GPR183 with EBNA1 and LMP1 was observed.

EBV Infection Induced GPR183 Expression in T and B Lymphocytes and Promoted IgG4 Production in B Lymphocytes

To further investigate the relationship among EBV infection, GPR183 expression, and the pathogenesis of IgG4-ROD, we analyzed the scRNA-seq dataset GSE189141, which contains B-lymphocyte enriched peripheral blood mononuclear cells collected before infection (day 0) and at days 2, 5, and 8 following in vitro EBV infection, with simultaneous detection of both human and EBV transcripts. Cell clustering and annotation were performed according to the original publication (Fig. 7A). Although the dataset was enriched for B lymphocytes, a small number of T lymphocytes and monocytes were also identified. Following EBV infection, GPR183 expression was upregulated in both B and T lymphocytes. In B lymphocytes, GPR183 expression peaked on day 2, whereas the highest expression level in T lymphocytes was observed on day 5. In parallel, the IGHG4/IGHG1 ratio in B lymphocytes progressively increased following EBV infection (Fig. 7C). To explore the mechanisms underlying EBV-induced GPR183 upregulation, we performed pseudotime analysis of B lymphocytes to reconstruct the differentiation trajectory following EBV infection (Fig. 7B). Notably, only six EBV genes (BKRF2, BVRF2, EBNA-3C, BALF1, BALF2, and BFLF1) reached their peak expression before GPR183, whereas most EBV genes peaked after GPR183 (Fig. 7D). Further analysis revealed that GPR183 expression reached its highest level during the activated precursor to early memory B-cell (AP-eMBC) stage (Figs. 7E, 7F).

Figure 7.

Single-cell RNA - seq analysis of lymphocytes before and after in vitro Epstein-Barr virus (EBV) infection. (A) Cell clustering and annotation performed according to the original publication. (B) Distribution of cells across infection time points and pseudotime trajectory analysis. (C) Expression levels of GPR183 in T lymphocytes and B lymphocytes, as well as the IGHG4/IGHG1 ratio in B lymphocytes at different time points. (D) Pseudotime positions at which EBV genes and GPR183 reached their peak expression, and their corresponding peak expression levels. (E) Dynamic expression pattern of GPR183 during B-lymphocyte differentiation. (F) GPR183 expression levels in different B-lymphocyte subsets at each time point.

Discussion

As a disease first defined in 2003, the pathogenesis of IgG4-RD remains elusive. IgG4-ROD, the most common subtype of IgG4-RD, can affect facial appearance and lead to diplopia or even irreversible vision loss. Currently, no disease-specific therapies exist for IgG4-ROD. High relapse rates and substantial adverse effects associated with systemic glucocorticoids represent the major clinical challenges in its management. These challenges underscore the urgent need to elucidate the upstream mechanisms driving IgG4-ROD in order to identify effective therapeutic targets.

In this study, we found that GPR183 was consistently and significantly upregulated across multiple IgG4-RD-affected tissues. Flow cytometry further demonstrated GPR183 expression in multiple immune cell populations within the lacrimal glands of patients with IgG4-ROD, including T lymphocytes, B lymphocytes, cDCs, and monocytes/macrophages. GPR183, also known as EBI2, binds ligands such as 7α,25-dihydroxycholesterol and other oxysterols, playing a critical role in germinal center formation.²⁰ In secondary lymphoid organs, such as the spleen and lymph nodes, stimulation of toll-like receptors (TLRs) on cDC2 upregulates GPR183 expression, driving cDC2 migration along oxysterol gradients toward the bridging channels in the spleen and the periphery of lymph node T-cell zones, which are sites of antigen accumulation and presentation.²¹ Concurrently, B lymphocytes and CD4+ T lymphocytes are recruited via GPR183 to the follicle-T zone interface, where they interact with activated cDC2s.²² Subsequently, cDC2s induce the differentiation of CD4+ T lymphocytes into Tfh, which in turn cooperate with B lymphocytes to establish germinal centers.²³ In this study, we observed prominent GPR183 expression within ectopic germinal centers in the lacrimal glands of patients with IgG4-ROD, particularly in B lymphocyte-rich regions. Collectively, these findings suggest that GPR183 may promote complex cellular interactions among diverse immune cell populations, thereby contributing to the formation and maintenance of ectopic germinal centers and facilitating B-lymphocyte differentiation in IgG4-ROD.

GPR183 was discovered in 1993 as an EBV-induced receptor in Burkitt lymphoma cell lines.¹⁹ EBV, also known as human herpesvirus 4 (HHV-4), is a double-stranded DNA virus that primarily infects children and adolescents, although adults may also be affected. Despite the fact that EBV is one of the most prevalent viruses worldwide, infecting approximately 90% of the population, the vast majority of infections remain asymptomatic.²⁴ After the EBV genome enters the cell nucleus, the virus can persist in the host by using complex mechanisms to evade immune detection and elimination, thereby causing no clinical symptoms.²⁵ However, during this process, EBV may trigger or exacerbate autoimmune diseases through mechanisms such as molecular mimicry and bystander activation. To date, EBV has been implicated in the pathogenesis of multiple autoimmune disorders, such as multiple sclerosis, rheumatoid arthritis, and Sjögren's syndrome.²⁶

Takeuchi et al.²⁷ and Furukawa et al.²⁸ have reported the presence of EBV in the lymph nodes and salivary glands of patients with IgG4-RD, providing preliminary evidence for a potential association between EBV and IgG4-RD. Because EBV is primarily transmitted through saliva, the salivary glands represent one of the initial sites of EBV infection, after which the virus establishes long-term latency in lymphocytes. Given that the salivary glands and lymph nodes are common sites of EBV infection, detecting EBV in these tissues offers limited power in establishing a link between EBV and IgG4-RD. In contrast, EBV detection in the lacrimal glands has been reported far less frequently. In this study, we first performed KEGG enrichment analysis using RNA-seq data and obtained preliminary indications that IgG4-ROD may be associated with EBV infection. We subsequently confirmed the presence of EBV in the lacrimal glands of patients with IgG4-ROD by qPCR and multiplex immunofluorescence. The infection rate was markedly higher in patients with IgG4-ROD than in controls, reaching 85.7%. Only 2 patients showed an EBV DNA copy number below 1 × 10³ copies/mL, which may reflect improper sample preservation leading to DNA degradation or a false-negative result from the detection kit. Moreover, the EBV DNA copy number in lacrimal gland tissues was positively correlated with serum IgG4 levels, further supporting the notion that EBV plays a significant role in the pathogenesis of IgG4-ROD. The consistent detection of EBV in the lymph nodes, salivary glands, and lacrimal glands of patients with IgG4-RD provides strong evidence supporting an association between EBV and IgG4-RD.

In addition, no previous studies have proposed potential mechanisms by which EBV may trigger or promote IgG4-RD. In our study, we found that the EBV infection markers EBNA1 and LMP1 were co-expressed with GPR183. This observation is consistent with earlier findings that EBV can induce upregulation of GPR183 in Burkitt lymphoma cell lines,¹⁹ supporting the possibility that EBV contributes to the pathogenesis of IgG4-ROD through GPR183 overexpression. To further investigate this possibility, we analyzed an scRNA-seq dataset derived from an in vitro EBV infection model of human lymphocytes. Our results demonstrated that EBV infection induced GPR183 upregulation in B lymphocytes and was accompanied by an increased IGHG4/IGHG1 ratio. Pseudotime analysis reconstructed the differentiation trajectory of B lymphocytes following EBV infection and revealed that only six EBV genes reached peak expression before GPR183. The rapid induction of GPR183 shortly after infection suggests that GPR183 may represent an early host response rather than a secondary consequence of extensive viral replication. Furthermore, GPR183 expression peaked during the transition from activated precursor B cells to early memory B cells, suggesting a role for GPR183 in early memory B-cell development and germinal-center-associated B-cell maturation. This observation is consistent with our multiplex immunofluorescence findings demonstrating prominent GPR183 expression within B lymphocytes enriched regions in ectopic germinal centers. Importantly, the IGHG4/IGHG1 ratio progressively increased following EBV infection, suggesting that EBV may promote class-switch recombination toward IgG4 production in B lymphocytes. Although the current data do not establish a direct causal relationship between GPR183 upregulation and IgG4 synthesis, these findings provide additional evidence supporting a link between EBV infection and IgG4-ROD. Interestingly, we also observed increased GPR183 expression in T lymphocytes following EBV infection. Although EBV infection of T lymphocytes has been reported, it is considerably less common than infection of B lymphocytes.²⁹ In our analysis, GPR183 expression peaked later in T lymphocytes than in B lymphocytes. This temporal difference may reflect the lower susceptibility of T lymphocytes to direct EBV infection. Alternatively, it may indicate that GPR183 upregulation in T lymphocytes is a secondary consequence of widespread immune activation rather than a direct effect of viral infection.

Several limitations of this study should be acknowledged. First, the relatively small sample size, together with potential demographic differences in age and sex between the patients with IgG4-ROD and control groups, may have influenced both GPR183 expression and EBV infection status. Consequently, the observed associations among EBV infection, GPR183 upregulation, and IgG4-ROD should be interpreted with caution and require validation in larger, well-matched independent cohorts. Second, whereas our data demonstrate a correlation between EBV and GPR183 expression, we cannot exclude alternative explanations. Local immune dysregulation in IgG4-ROD might facilitate EBV reactivation, or inflammatory cytokines could independently drive GPR183 upregulation. Therefore, future studies utilizing an EBV-induced animal model of IgG4-ROD are essential to directly establish the causal relationship among EBV infection, GPR183 upregulation, and disease pathogenesis. Nevertheless, our findings provide important guidance for future research directions. Although IgG4-ROD is generally regarded as a non-infectious autoimmune disorder, it remains worth investigating whether infectious factors—particularly EBV—play a contributory role.

Conclusions

This study revealed a potential link between EBV infection and IgG4-ROD and suggests that EBV may contribute to disease pathogenesis through upregulation of GPR183 expression. This discovery offers an alternative perspective to the traditional view that IgG4-ROD is a non-infectious autoimmune disease and provides new directions and perspectives for research into its pathogenesis, diagnostic approaches, and therapeutic targets.