A STING inhibitor suppresses EBV-induced B cell transformation and lymphomagenesis

Abstract

Epstein-Barr virus-associated lymphoproliferative disease (EBV-LPD) is frequently fatal. Innate immunity plays a key role in protecting against pathogens and cancers. The stimulator of interferon genes (STING) is regarded as a key adaptor protein allowing DNA sensors recognizing exogenous cytosolic DNA to activate the type I interferon signaling cascade. In terms of EBV tumorigenicity, the role of STING remains elusive. Here we showed that treatment with the STING inhibitor, C-176, suppressed EBV-induced transformation in peripheral blood mononuclear cells. In an EBV-LPD mouse model, C-176 treatment also inhibited tumor formation and prolonged survival. Treatment with B cells alone did not affect EBV transformation, but suppression of EBV-induced transformation was observed in the presence of T cells. Even without direct B cell-T cell contact in a transwell system, the inhibitor reduced the transformation activity, indicating that intercellular communication by humoral factors was critical to prevent EBV-induced transformation. These findings suggest that inhibition of STING signaling pathway with C-176 could be a new therapeutic target of EBV-LPD.

Keywords: EBV-LPD; Epstein-Barr virus; PAMPs; STING; cGAS.

FIGURE 1

STING inhibitor suppresses EBV tumor formation in the EBV‐LPD mouse model. A, Schematic of the mouse model. A NOG mouse was inoculated ip with EBV‐infected human cord blood mononuclear cells and then administered DMSO or C‐176 (562.5 nmol) ip, twice daily for 2 wk (n = 6 mice per group). Blood sampling was carried out to identify genomic EBV DNA at the indicated time points. B, Kaplan‐Meier survival curves. C, Bodyweight changes of mice are shown. D, The ip tumor and spleen of DMSO‐treated and C‐176‐treated mice. The EBV‐encoded EGFP signal was detected under excitation light. Scale bars, 5 mm. E, The EBV genome (copies/mL) of each group is indicated. EBV DNA in blood was detected by real‐time PCR. n.s., no significant difference. F, H&E staining of DMSO‐treated and C‐176‐treated mouse organs. Scale bars, 200 μm

FIGURE 2

An inhibitor of STING suppresses EBV transformation in vitro but not STING signaling in EBV‐positive B cells. A, Primary B cells and EBV‐transformed B cells, known as LCLs and established from several healthy donor‐derived PBMCs, were analyzed by immunoblotting for STING. Epstein‐Barr virus LMP1 was used as an infection marker. Anti‐GAPDH antibody was used as the internal control. B, PBMCs were infected with 10‐fold serial dilutions of Akata EBV. The medium containing the C‐176 STING inhibitor (0.5 µM) was exchanged every 4 d. After 3 wk, the transformation efficiency (TD₅₀/mL) was calculated based on the number of wells in which immortalized cells were present. C, LCLs were cultured in the presence of C‐176 and counted every 24 h for 4 d. A trypan blue exclusion test using an automated cell counter was performed. D, LCLs were treated with the indicated concentration of C‐176 for 24 h, and cell viability was quantified by a luminescent assay using CellTiter‐Glo, based on the ATP level. E, Cytokine production of EBV‐transformed B cells treated with DMSO or STING inhibitor (C‐176). LCLs treated with C‐176 for 24 h were harvested and total RNA was extracted. IFN‐β, IL‐6, and CXCL10 mRNAs were quantified by RT‐PCR. All experiments were carried out in triplicate. n.s., no significant difference, Student t test. F, Immunoblot analysis of STING, TBK1, phospho‐TBK1 (Ser 172), and GAPDH of LCLs, in the presence or absence of C‐176

FIGURE 3

Tumor‐infiltrating T cells express STING. A, H&E and immunohistochemical staining for STING, CD20, and CD3, and in situ hybridization for EBER in tumor tissue from the EBV‐LPD model mouse. Scale bars, 50 μm. The black boxes contain magnified images. B, Co‐immunofluorescence staining for STING (green), LMP1 (magenta), and DAPI (gray) in the tumor of the EBV‐LPD mouse model. C, The relative proportions of CD45‐, CD3‐, CD8‐, CD19‐, and CD56‐positive cells in splenic lymphocytes of the EBV‐LPD mouse model were determined by flow cytometry. D, Co‐immunofluorescence staining of STING (green), CD3 (magenta), and DAPI (cyan) in the tumor of the EBV‐LPD mouse model. Scale bars, 50 μm. The white boxes contain magnified images. E, Flow cytometric analysis of STING in splenic lymphocytes in the EBV‐LPD mouse model

FIGURE 4

T cells promote EBV transformation through humoral factors. A, B cells isolated from PBMCs were infected with Akata EBV and cultured for 3 wk in medium containing DMSO or C‐176 (0.5 µM). Wells with transformed cells were counted within a 96‐well plate to calculate the EBV transformation efficiency (TD₅₀/mL). This assay was also performed under B and T cell co‐culture conditions. B, Schematic of the transwell system. Isolated B cells were seeded into a 24‐well plate and cultured with medium containing C‐176 (0.5 µM), while isolated T cells were inoculated into transwell chambers with 0.4 µm polycarbonate membranes. Cells were co‐cultured for 7 d and observed by fluorescence microscopy. Scale bard, 200 μm

FIGURE 5

STING inhibitors suppressed STING signaling in T cells. Immunoblot analysis showing STING, TBK1, phospho‐TBK1 (Ser172), and GAPDH. A, B, Primary T cells were isolated from PBMCs and reacted with 0.5 µM C‐176 or 0.5 µM H‐151 for 24 h. C, Primary T cells were exposed to DMSO or 50 µM G10 for 2 h. D, Jurkat cells were treated with DMSO or 50 µM G10 and harvested at the indicated times. E, Human CBMC‐derived B cells were co‐cultured with EBV in vitro. On day 1 after infection, expanded T cells carrying shScramble or shSTING (#1 and #2) were seeded into the transwell chamber. F, Immunoblots for endogenous STING in shSTING‐transduced T cells. G, Fluorescence image of B cells co‐cultured with T cells. Scale bars, 200 μm. H, B cells were quantified at 7 d after infection by counting trypan blue‐stained cells