Induction of EBV latent membrane protein-2A (LMP2A)-specific T cells and construction of individualized TCR-engineered T cells for EBV-associated malignancies

BackgroundLatent membrane protein-2A (LMP2A)-specific TCR-engineered T cells could be a promising treatment approach to Epstein–Barr virus-associated malignancies. However, previous studies mainly reported LMP2A-reactive TCRs only focusing on specific HLA subtypes and corresponding epitopes, and thus, they were only suitable for patients with specific HLA.MethodsDue to hugely varied HLA subtypes and presented LMP2A epitopes in different individuals, our study attempted to develop an individualized approach, based on the weekly in vitro stimulation of peripheral T cells for 2 weeks with autologous dendritic cells (DCs) pulsed with a pool of LMP2A peptides covering LMP2A whole protein and combination analysis of high throughput TCRβ sequencing of prestimulated and poststimulated T cells and single-cell TCR sequencing of poststimulated T cells, and to identify LMP2A-specific TCRs of which poststimulated frequencies significantly increased than corresponding prestimulated frequencies.ResultsFollowing this approach, multiple LMP2A-reactive TCRs were identified, optimized and cloned into lentiviral vector, and then transduced into peripheral T cells. These engineerd T cells were demonstrated to specifically recognize the LMP2A presented by autologous DCs and lymphoblastoid cell lines in vitro and in vivo.ConclusionsThis approach provides an efficient procedure to isolate individualized LMP2A-specific TCRs for basic and translational research, as well as for clinical applications.

Epstein-Barr Virus-Encoded Latent Membrane Protein 2A Downregulates GCNT3 via the TGF-β1/Smad-mTORC1 Signaling Axis

Increasing evidence shows that Epstein-Barr virus (EBV) infection is closely related to various lymphoid and epithelioid malignancies. However, the underlying mechanisms are unclear. GCNT3 (core 2β-1,6-acetylglucosaminyltransferase) is a new type of core mucin synthase, and its expression in EBV-associated gastric cancer (EBVaGC) is lower than that in EBV-negative gastric cancer (EBVnGC). EBV-encoded latent membrane protein 2A (LMP2A) is a transmembrane protein with tumorigenic transformation properties. Here, we demonstrated that LMP2A inhibited the transcription of GCNT3 by inhibiting Smad2/3 and Smad4. LMP2A restrained the activation of the mTORC1 pathway by inactivating the TGF-β1/Smad pathway and then downregulated GCNT3 expression. The mTORC1-GCNT3 pathway promoted cell proliferation and migration and inhibited G0/G1 cell arrest. Related proteins involved in epithelial-mesenchymal transition (EMT) were downstream molecules of the TGF-β1/Smad-mTORC1-GCNT3 pathway. GCNT3 inhibited autophagy by inducing mTORC1 phosphorylation. These findings indicate that targeting the TGF-β1/Smad-mTORC1-GCNT3 axis may represent a novel therapeutic target in GC. Importance Epstein-Barr virus (EBV) is an opportunistic pathogen, and the latent membrane protein 2A (LMP2A) encoded by EBV plays a key role in ensuring the incubation period of EBV. Glycosylation modification is an important marker of cancer cells, and recent studies have reported that it is related to EBV. Our conclusions provide deeper theoretical support for the role of LMP2A and TGF/Smad-mTORC1-GCNT3 in EBVaGC and help to understand glycosylation abnormalities in cancer. Our results may provide novel therapeutic targets for the treatment of gastric cancer against the TGF/Smad-mTORC1-GCNT3 signaling cascade.

The impact of EBV on the epigenetics of gastric carcinoma

EBV is an important human tumor virus and is closely related to the occurrence of a variety of tumors, involving 10% of gastric cancer. In EBV-associated gastric carcinoma (EBVaGC), EBV expresses restrict viral genes including EBV nuclear antigen 1, EBV encoded small RNAs, Bam HI-A rightward transcripts, latent membrane protein 2A and miRNAs. The role of EBV in gastric carcinogenesis has received increasing attention and is considered to be another pathogenic factor in addition to Helicobacter pylori. A typical characteristic of EBVaGC is the extensive methylation of viral and host genome. Combined with other epigenetic mechanisms, EBV infection acts as an epigenetic driver of EBVaGC oncogenesis. In this review we discuss recent findings of EBV effect on host epigenetic alterations in EBVaGC and its role in oncogenic process.

Immune Landscape in Burkitt Lymphoma reveals M2-macrophage polarization and Correlation between PD-L1 expression and Non-Canonical EBV latency program

BACKGROUND: The Tumor Microenviroment (TME) is a complex milieu that is increasingly recognized as a key factor in multiple stages of disease progression and responses to therapy as well as escape from immune surveillance. However, the precise contribution of specific immune effector and immune suppressor components of the TME in Burkitt lymphoma (BL) remains poorly understood.METHODS: In this paper, we applied the computational algorithm CIBERSORT to Gene Expression Profiling (GEP) datasets of 40 BL samples to draw a map of immune and stromal components of TME. Furthermore, by multiple immunohistochemistry (IHC) and multispectral immunofluorescence (IF), we investigated the TME of additional series of 40 BL cases to evaluate the role of the Programmed Death-1 and Programmed Death Ligand-1 (PD-1/PD-L1) immune checkpoint axis. RESULTS: Our results indicate that M2 polarized macrophages are the most prominent TME component in BL. In addition, we investigated the correlation between PD-L1 and latent membrane protein-2A (LMP2A) expression on tumour cells, highlighting a subgroup of BL cases characterized by a non-canonical latency program of EBV with an activated PD-L1 pathway. CONCLUSION: In conclusion, our study analysed the TME in BL and identified a tolerogenic immune signature highlighting new potential therapeutic targets.

Immune Landscape in Burkitt Lymphoma reveals M2-macrophage polarization and Correlation between PD-L1 expression and Non-Canonical EBV latency program

BACKGROUND: The Tumor Microenviroment (TME) is a complex milieu that is increasingly recognized as a key factor in multiple stages of disease progression and responses to therapy as well as escape from immune surveillance. However, the precise contribution of specific immune effector and immune suppressor components of the TME in Burkitt lymphoma (BL) remains poorly understood.METHODS: In this paper, we applied the computational algorithm CIBERSORT to Gene Expression Profiling (GEP) datasets of 40 BL samples to draw a map of immune and stromal components of TME. Furthermore, by multiple immunohistochemistry (IHC) and multispectral immunofluorescence (IF), we investigated the TME of additional series of 40 BL cases to evaluate the role of the Programmed Death-1 and Programmed Death Ligand-1 (PD-1/PD-L1) immune checkpoint axis. RESULTS: Our results indicate that M2 polarized macrophages are the most prominent TME component in BL. In addition, we investigated the correlation between PD-L1 and latent membrane protein-2A (LMP2A) expression on tumour cells, highlighting a subgroup of BL cases characterized by a non-canonical latency program of EBV with an activated PD-L1 pathway. CONCLUSION: In conclusion, our study analysed the TME in BL and identified a tolerogenic immune signature highlighting new potential therapeutic targets.

Immune Landscape in Burkitt Lymphoma Reveals M2-Macrophage Polarization and Correlation between PD-L1 Expression and Non-Canonical EBV Latency Program

The Tumor Microenviroment (TME) is a complex milieu that is increasingly recognized as a key factor in multiple stages of disease progression and responses to therapy as well as escape from immune surveillance. However, the precise contribution of specific immune effector and immune suppressor components of the TME in Burkitt lymphoma (BL) remains poorly understood. In this paper, we applied the computational algorithm CIBERSORT to Gene Expression Profile (GEP) datasets of 40 BL samples to draw a map of immune and stromal components of TME. Furthermore, by VECTRA multispectral immunofluorescence (IF) and multiple immunohistochemistry (IHC), we investigated the TME of an additional series of 40 BL cases and evaluated the possible role of the PD-1/PD-L1 immune checkpoint axis. Our results indicated that M2 polarized macrophages are the most prominent TME component in BL. In addition, we investigated the correlation between PD-L1 and latent membrane protein-2A (LMP2A) expression on tumour cells, highlighting a subgroup of BL cases characterized by a non- canonical latency program of EBV with an activated PD-L1 pathway. In conclusion, our study analysed the TME in BL and identified a tolerogenic immune signature highlighting new potential therapeutic targets.

Two Pathways of p27Kip1Degradation Are Required for Murine Lymphoma Driven by Myc and EBV Latent Membrane Protein 2A

ABSTRACTEpstein-Barr virus (EBV) latent membrane protein 2A (LMP2A), expressed in EBV latency, contributes to Burkitt lymphoma (BL) development in a murine model by acting as a constitutively active B cell receptor (BCR) mimic. Mice expressing both LMP2A andMYCtransgenes (LMP2A/λ-MYC) develop tumors significantly faster than mice only expressingMYC(λ-MYC). Previously, we demonstrated the cell cycle inhibitor p27Kip1is present at significantly lower levels in LMP2A/λ-MYCmice due to increased posttranslational degradation. P27Kip1degradation can occur in the cytoplasm following phosphorylation on serine 10 (S10) or in the nucleus via the SCFSkp2complex, which depends on Cks1. We previously demonstrated an S10A knock-in of p27Kip1(p27S10A/S10A) that prevented S10 phosphorylation failed to significantly delay tumor onset in LMP2A/λ-MYCmice. We also previously demonstrated that aCks1knockout partially delayed tumor onset in LMP2A/λ-MYCmice, but onset was still significantly faster than that in λ-MYCmice. Here, we have combined both genetic manipulations in what we call p27Supermice. LMP2A/λ-MYC/p27Supermice and λ-MYC/p27Supermice both displayed dramatic delays in tumor onset. Strikingly, tumor development in LMP2A/λ-MYC/p27Supermice was later than that in λ-MYCmice and not significantly different from that in λ-MYC/p27Supermice. The p27Supergenotype also normalized G1-S-phase cell cycle progression, spleen size, and splenic architecture in LMP2A/λ-MYCmice. Our results reveal both major pathways of p27Kip1degradation are required for the accelerated BL development driven by LMP2A in our BL model and that blocking both degradation pathways is sufficient to delay Myc-driven tumor development with or without LMP2A.IMPORTANCEBL is a cancer that primarily affects children. The side effects of chemotherapy highlight the need for better BL treatments. Many BL tumors contain EBV, and our goal is to determine what makes EBV-positive BL different from EBV-negative BL. This may lead to more specific treatments for both types. All cases of BL require overexpression ofMYC. Mice engineered to express EBV LMP2A along withMYC(LMP2A/λ-MYCmice) develop tumors much more quickly than mice only expressingMYC(λ-MYCmice). Blocking degradation of the cell cycle inhibitor protein p27Kip1in LMP2A/λ-MYCmice causes tumors to develop later than in λ-MYCmice, showing that p27Kip1degradation may play a larger role in EBV-positive BL than EBV-negative BL. Furthermore, our studies suggest the cell cycle is an attractive target as a treatment option for LMP2A-positive cancers in humans.

Two pathways of p27Kip1degradation are required for murine lymphoma driven by Myc and EBV latent membrane protein 2A

Epstein-Barr virus (EBV) latent membrane protein 2A (LMP2A), expressed in EBV latency, contributes to Burkitt Lymphoma (BL) development in a murine model by acting as a constitutively active B cell receptor (BCR) mimic. Mice expressing both LMP2A andMYCtransgenes (LMP2A/λ-MYC) develop tumors significantly faster than mice only expressingMYC(λ-MYC). Previously, we demonstrated the cell cycle inhibitor p27Kip1is present at significantly lower levels in LMP2A/λ-MYCmice due to increased post-translational degradation. P27Kip1degradation can occur in the cytoplasm following phosphorylation on serine 10 (S10), or in the nucleus via the SCFSkp2complex, which depends on Cks1. We previously demonstrated a S10A knock-in of p27Kip1(p27S10A/S10A), which prevented S10 phosphorylation, failed to significantly delay tumor onset in LMP2A/λ-MYCmice. We also previously demonstrated that aCks1knockout partially delayed tumor onset in LMP2A/λ-MYCmice, but onset was still significantly faster than in λ-MYCmice. Here, we have combined both genetic manipulations in what we call p27Supermice. LMP2A/λ-MYC/p27Supermice and λ-MYC/p27Supermice both displayed dramatic delays in tumor onset. Strikingly, tumor development in LMP2A/λ-MYC/p27Supermice was later than in λ-MYCmice and not significantly different from λ-MYC/p27Supermice. The p27Supergenotype also normalized G1-S phase cell cycle progression, spleen size, and splenic architecture in LMP2A/λ-MYCmice. Our results reveal both major pathways of p27Kip1degradation are required for the accelerated BL development driven by LMP2A in our BL model and that blocking both degradation pathways is sufficient to delay Myc-driven tumor development with or without LMP2A.ImportanceBurkitt lymphoma (BL) is a cancer that primarily affects children. The side effects of chemotherapy highlight the need for better BL treatments. Many BL tumors contain Epstein-Barr virus (EBV) and our goal is to determine what makes EBV-positive BL different from EBV-negative BL. This may lead to more specific treatments for both types. All cases of BL require overexpression ofMYC. Mice engineered to express an EBV LMP2A along withMYC(LMP2A/λ-MYCmice) develop tumors much more quickly than mice only expressing MYC (λ-MYCmice). Blocking degradation of the cell cycle inhibitor protein p27Kip1in LMP2A/λ-MYCmice causes tumors to develop later than in λ-MYCmice, showing that p27Kip1degradation may play a larger role in EBV-positive BL than EBV-negative. Furthermore, our studies suggest the cell cycle may be an attractive target as a treatment option for LMP2A positive cancers in humans.