Imbalanced Expression of TACI Isoforms Regulated By TNFRSF13B Variants Promotes the Progression of Epstein-Barr Virus-Associated Lymphoproliferative Diseases

INTRODUCTION: Epstein-Barr virus (EBV) is a commonly distributed herpes virus. EBV usually infects B and epithelial cells, but it is also able to infect T and natural killer (NK) cells and trigger the intractable EBV-associated T/NK lymphoproliferative diseases (EBV-T/NK-LPDs) in rare cases. There exists a large number of patients without any pathogenic gene variants despite several genetic defects such as those in UNC13D and LYST being identified in primary EBV-T/NK-LPDs. In the authors’ previous retrospective study, the germline tumor necrosis factor (TNF)-TNF receptor (TNFR) superfamily members variants were recurrently found in non-primary EBV-T/NK-LPDs patients, with the TNFRSF13B exon 2 variants being the most frequent. The signaling interactions between TACI (encoded by TNFRSF13B) and EBV-encoded proteins, and the physiological process shared by EBV lytic reactivation and plasma cell differentiation, strongly indicate that TACI variants alter the anti-EBV responses in multiple aspects. In this study, we revealed the imbalanced expression of TACI isoforms caused by distinct TACI variants, investigated the possible mechanisms of TACI variants-mediated alterations of EBV activity, and discussed the possible TACI-targeted therapies for EBV-T/NK-LPDs. This preliminary research provided a novel future direction for decoding EBV infection and anti-EBV immunity.

METHODS: To investigate the role of recurrent TNFRSF13B variants in the process of EBV infection and immortalization, B lymphoblastoid cell lines (B-LCL) models carrying homozygous TNFRSF13B exon 2 frameshift deletions/insertions was successfully constructed using the CRISPR/Cas9 technique. Several examinations are performed to clarify the influences of TACI variants in EBV-infected B cells, including the TACI expression, cell viability, B-lineage immunophenotype, the activation of downstream signaling pathways, the EBV lytic replication/latency program, and the secretion of cytokines. To verify the potentials of TNFRSF13B-targeting therapy in EBV-LPDs, drugs that regulate TACI expression or the TACI-mediated signaling were applied in B-LCL models. Finally, the possible mechanisms of TACI-mediated EBV reactivation in the immortalized B cells were investigated primarily through transcriptomic analysis and subsequent validation experiments.

RESULTS: In the cohort of previous research, six non-primary EBV-T/NK-LPD patients carry rare heterozygous pathogenic germline TNFRSF13B variants, of which 5 mutations were located at the exon 2 of this gene. Introducing frameshift deletions/insertions at the exon 2 of TNFRSF13B in healthy donor-derived B-LCLs diminished the expression of long isoforms of TACI (TACI-L) but significantly upregulated the short isoforms of TACI (TACI-S). The enhanced expression of TACI-S delivered more intense activation of NF-κB, MAPK, Rho, and PI3K/AKT pathways, induced the reactivation of EBV lytic replication in LCLs, promoted the secretion of viral interleukin (IL)-10 and pro-inflammatory cytokines, ultimately led to the significantly increased EBV loads, rendering the carriers susceptible to EBV-LPDs. Furthermore, the low-affinity 3-mer forms of TACI ligand, B cell-activating factor (BAFF), and a proliferation-inducing ligand (APRIL) significantly reduced TACI signaling, suppressing the EBV lytic program, and thus alleviating the EBV infection in B-LCLs. The AKT signaling suppressor TUSC3 differently expressed in TACI exon 2 variant-expressing and wild-type B-LCLs, might mediate the TACI-associated EBV lytic reactivation.

CONCLUSIONS: This research proposes that the TNFRSF13B variants play important roles during the process of EBV-LPD development by regulating the expression of TACI isoforms. For patients who carry the gain-of-function germline mutations of TNFRSF13B, the inhibitory TACI-Ig or the TACI ligands BAFF and APRIL may efficiently prevent the serious progression of their EBV infection. Screening for possibly pathogenic variants, especially in the TNF-TNFR superfamily genes in EBV-LPD patients is necessary and valuable for understanding the pathogenesis of EBV-LPDs and optimizing the therapeutic regimen.