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

2021 ◽  
Vol 9 (7) ◽  
pp. e002516
Author(s):  
Chaoting Zhang ◽  
Qin Tan ◽  
Shance Li ◽  
Luyan Shen ◽  
Jingtao Zhang ◽  
...  
Keyword(s):  
T Cells ◽  
Membrane Protein ◽  
Lentiviral Vector ◽  
Latent Membrane Protein ◽  
Latent Membrane Protein 2A ◽  
Barr Virus ◽  
Peripheral T Cells ◽  
Engineered T Cells

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.

scholarly journals Role of Epstein–Barr virus-encoded latent membrane protein 2A on virus-induced immortalization and virus activation

2001 ◽  
Vol 82 (6) ◽  
pp. 1451-1456 ◽  
Author(s):  
Kazuya Konishi ◽  
Seiji Maruo ◽  
Hiroyuki Kato ◽  
Kenzo Takada
Keyword(s):  
Membrane Protein ◽  
B Lymphocytes ◽  
Virus Replication ◽  
Epstein Barr Virus ◽  
Latent Membrane Protein ◽  
Latent Membrane Protein 2A ◽  
Barr Virus ◽  
Epstein Barr

To quantitatively evaluate the role of Epstein-Barr virus (EBV)-encoded latent membrane protein 2A (LMP2A) in immortalization of peripheral B-lymphocytes, we used the Akata cell system to generate an EBV recombinant in which the first exon of the LMP2A gene was disrupted. The results indicated that deletion of the LMP2A gene did not affect the immortalization efficiency of EBV in B-lymphocytes. Deletion of the LMP2A gene made EBV-transformed lymphocytes more permissive for virus replication in response to surface immunoglobulin cross-linking. On the other hand Akata cells, in which LMP2A expression was much lower than in EBV-transformed lymphocytes, were equally permissive for virus replication whether they were infected with wild EBV or LMP2A-knockout EBV. The results raise a question as to the role of LMP2A in inhibition of disruption of virus latency in vivo, where LMP2A expression has been expected to be low as in Akata cells.

scholarly journals Epstein-Barr Virus Latent Membrane Protein 2A Preferentially Signals through the Src Family Kinase Lyn

2008 ◽  
Vol 82 (17) ◽  
pp. 8520-8528 ◽  
Author(s):  
Mark Rovedo ◽  
Richard Longnecker
Keyword(s):  
B Cells ◽  
Membrane Protein ◽  
B Cell ◽  
Epstein Barr Virus ◽  
Sh2 Domain ◽  
Latent Membrane Protein ◽  
Latent Membrane Protein 2A ◽  
Barr Virus ◽  
Epstein Barr

ABSTRACT Latent membrane protein 2A (LMP2A) is a viral protein expressed during Epstein-Barr virus (EBV) latency in EBV-infected B cells both in cell culture and in vivo. LMP2A has important roles in modulating B-cell receptor signal transduction and provides survival and developmental signals to B cells in vivo. Although Lyn has been shown to be important in mediating LMP2A signaling, it is still unclear if Lyn is used preferentially or if LMP2A associates promiscuously with other Src family kinase (SFK) members. To investigate the role of various SFKs in LMP2A signaling, we crossed LMP2A transgenic mice (TgE) with Lyn−/−, Fyn−/−, or Blk−/− mice. TgE Lyn−/− mice had a larger immunoglobulin M (IgM)-positive B-cell population than TgE mice, suggesting that the absence of Lyn prevents LMP2A from delivering survival and developmental signals to the B cells. Both TgE Fyn−/− and TgE Blk−/− mice have an IgM-negative population of splenic B cells, similar to the TgE mice. LMP2A was also transiently transfected into the human EBV-negative B-cell line BJAB to determine which SFK members associate with LMP2A. Lyn was detected in LMP2A immunoprecipitates, whereas Fyn was not. Both Lyn and Fyn were able to bind to an LMP2A mutant which contained a sequence shown previously to bind tightly to the SH2 domain of multiple SFK members. From these results, we conclude that LMP2A preferentially associates with and signals through Lyn compared to its association with other SFKs. This preferential association is due in part to the SH2 domain of Lyn associating with LMP2A.

scholarly journals Hematopoietic lineage-converted T cells carrying tumor-associated antigen-recognizing TCRs effectively kill tumor cells

2020 ◽  
Vol 8 (2) ◽  
pp. e000498
Author(s):  
Fangxiao Hu ◽  
Dehao Huang ◽  
Yuxuan Luo ◽  
Peiqing Zhou ◽  
Cui Lv ◽  
...  
Keyword(s):  
T Cells ◽  
B Cells ◽  
Tumor Cells ◽  
Cell Receptor ◽  
Histocompatibility Complex ◽  
Tumor Associated Antigen ◽  
Engineered T Cells ◽  
Antitumor Immunotherapy

Tumor-associated antigen (TAA) T-cell receptor (TCR) gene-engineered T cells exhibit great potential in antitumor immunotherapy. Considering the high costs and low availability of patient-derived peripheral blood T cells, substantial efforts have been made to explore alternatives to natural T cells. We previously reported that enforced expression of Hoxb5 converted B cells into induced T (iT) cells in vivo. Here, we successfully regenerated naive OT1 (major histocompatibility complex I restricted ovalbumin antigen) iT cells (OT1-iT) in vivo by expressing Hoxb5 in pro-pre-B cells in the OT1 transgenic mouse. The OT1-iT cells can be activated and expanded in vitro in the presence of tumor cells. Particularly, these regenerated OT1-iT cells effectively eradicated tumor cells expressing the TAA (ovalbumin) both in vitro and in vivo. This study provides insights into the translational applications of blood lineage-transdifferentiated T cells in immunotherapy.

scholarly journals In vitro-transcribed antigen receptor mRNA nanocarriers for transient expression in circulating T cells in vivo

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
N. N. Parayath ◽  
S. B. Stephan ◽  
A. L. Koehne ◽  
P. S. Nelson ◽  
M. T. Stephan
Keyword(s):  
T Cells ◽  
Ex Vivo ◽  
Human Leukemia ◽  
Antigen Receptors ◽  
Wide Range ◽  
Engineered T Cells ◽  
Circulating T Cells ◽  
The Cost

AbstractEngineering chimeric antigen receptors (CAR) or T cell receptors (TCR) helps create disease-specific T cells for targeted therapy, but the cost and rigor associated with manufacturing engineered T cells ex vivo can be prohibitive, so programing T cells in vivo may be a viable alternative. Here we report an injectable nanocarrier that delivers in vitro-transcribed (IVT) CAR or TCR mRNA for transiently reprograming of circulating T cells to recognize disease-relevant antigens. In mouse models of human leukemia, prostate cancer and hepatitis B-induced hepatocellular carcinoma, repeated infusions of these polymer nanocarriers induce sufficient host T cells expressing tumor-specific CARs or virus-specific TCRs to cause disease regression at levels similar to bolus infusions of ex vivo engineered lymphocytes. Given their ease of manufacturing, distribution and administration, these nanocarriers, and the associated platforms, could become a therapeutic for a wide range of diseases.

scholarly journals Epstein-Barr virus latent membrane protein 2A exploits Notch1 to alter B-cell identity in vivo

Blood ◽  
2009 ◽  
Vol 113 (1) ◽  
pp. 108-116 ◽  
Author(s):  
Leah J. Anderson ◽  
Richard Longnecker
Keyword(s):  
Membrane Protein ◽  
B Cell ◽  
Epstein Barr Virus ◽  
Cell Development ◽  
B Cell Development ◽  
Latent Membrane Protein ◽  
Cell Identity ◽  
Barr Virus ◽  
Epstein Barr

Abstract Expression of latent membrane protein 2 (LMP2A) during B-cell development leads to global alterations in gene transcription similar to those seen in Hodgkin Reed-Sternberg cells of Hodgkin lymphoma (HL). Along with the consistent detection of LMP2A in Epstein-Barr virus–associated HL, this implicates a role for LMP2A in the pathogenesis of HL. We have shown that LMP2A constitutively activates the Notch1 pathway to autoregulate the LMP2A promoter. To determine whether constitutive activation of the Notch pathway is important for LMP2A-mediated alterations in B-cell development in vivo, TgE-LMP2A–transgenic mice were intercrossed with mice expressing loxP-flanked Notch1 genes and Cre recombinase. B cells from TgE Notch1lox/lox-CD19+/Cre mice have an increase in immunoglobulin M and CD43 and a decrease in CD5 expression in the bone marrow compared with TgE Notch1lox/lox mice, indicating the LMP2A signal for developmental aberrations is impaired in the absence of Notch1. Real-time reverse-transcribed polymerase chain reaction analysis reveals that LMP2A requires the Notch1 pathway to alter levels of B cell–specific transcription factors, E2A and EBF. Interestingly, Notch1 appears to be important for LMP2A-mediated survival in low interleukin-7. We propose that LMP2A and the Notch1 pathway may cooperate to induce the alterations in B-cell identity seen in Hodgkin Reed-Sternberg cells.

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