Measurement of CD8+ and CD4+ T Cell Frequencies Specific for EBV LMP1 and LMP2a Using mRNA-Transfected DCs

Abstract

An EBV-specific cellular immune response is associated with the control of EBV-associated malignancies and lymphoproliferative diseases, some of which have been successfully treated by adoptive T cell therapy. Therefore, many methods have been used to measure EBV-specific cellular immune responses. Previous studies have mainly used autologous EBV-transformed B-lymphoblastoid cell lines (B-LCLs), recombinant viral vectors transfected or peptide pulsed dendritic cells (DCs) as stimulators of CD8+ and CD4+ T lymphocytes. In the present study, we used an interferon-γ (IFN-γ) enzyme-linked immunospot (ELISPOT) assay by using isolated CD8+ and CD4+ T cells stimulated with mRNA-transfected DCs. The frequency of latent membrane protein 1 (LMP1)-specific IFN-γ producing CD4+ T cells was significantly higher than that of LMP2a. The frequency of IFN-γ producing CD4+ T cells was significantly correlated with that of CD8+ T cells in LMP1-specific immune responses (r = 0.7187, Pc < 0.0001). To determine whether there were changes in LMP1- or LMP2a-specific immune responses, subsequent peripheral blood mononuclear cells (PBMCs) samples were analyzed. Significant changes were observed in 5 of the 10 donors examined, and CD4+ T cell responses showed more significant changes than CD8+ T cell responses. CD8+ and CD4+ T cells from EBV-seropositive donors secreted only the Th1 cytokines IFN-γ, TNF-α, and IL-2, while Th2 (IL-4) and Th17 (IL-17a) cytokines were not detected. CD4+ T cells secreted significantly higher cytokine levels than did CD8+ T cells. Analysis of EBV-specific T cell responses using autologous DCs transfected with mRNA might provide a comprehensive tool for monitoring EBV infection and new insights into the pathogenesis of EBV-associated diseases.

Comprehensive Analysis of Cytomegalovirus pp65 Antigen-Specific CD8+ T Cell Responses According to Human Leukocyte Antigen Class I Allotypes and Intraindividual Dominance

“…The IFN-γ ELISPOT assay was performed as described previously in literature, with modifications ( 19 ). Briefly, autologous CD8 + T cells (10 6 cells) were serially diluted in complete RPMI and 100 µL of each cell suspension was then transferred into the wells, followed by the addition of 200 µL of aAPC and aAPC-pp65 (10 5 cells) untransfected or transfected with individual HLA-matched single HLA allotype.…”

“…ELISPOT using pp65-transduced CD40-activated B cells has been used for identifying CTL epitopes presented by various HLA allotypes ( 10 ). Epstein–Barr virus (EBV)-specific CD8 + T cell responses can be evaluated using autologous dendritic cells transfected with EBV latent membrane protein 1 and latent membrane protein 2A mRNA ( 19 ). To comprehensively analyze CD8 + T cell responses against the CMV pp65 antigen restricted by a single HLA class I allotype, we conducted ELISPOT assays using an artificial antigen-presenting cell (aAPC) expressing both the pp65 antigen and each HLA class I allotype present in a donor.…”

NF-κB Pathways in the Pathogenesis of Multiple Sclerosis and the Therapeutic Implications

“…However, the persistent antigen presence of EBV-infected B-blasts triggers a robust T cell response that controls the events of reactivation (Hislop et al, 2005 ; Lünemann et al, 2007 ; Lassmann et al, 2011 ). The mechanism may involve LMP1 as it can act on T cells and studies have shown the responses are particularly T H 1 polarized leading to IFN-γ, TNF-α and IL-2 secretion (Sohn et al, 2015 ).…”

Comprehensive Analysis of Epstein-Barr Virus LMP2A-Specific CD8+ and CD4+ T Cell Responses Restricted to Each HLA Class I and II Allotype Within an Individual

“…In our previous study, CD8 + and CD4 + T cells responding to LMP1 and LMP2A, respectively, were measured in mRNA-transfected dendritic cells (DCs). As a result, we observed that the LMP1-specific CD4 + T cell response was significantly higher than the LMP2A-specific CD4 + T cell response ( 5 ).…”

“…EBV is associated with human malignancies, including Burkitt's lymphoma, Nasopharyngeal carcinoma, Hodgkin’s lymphoma, and lymphoproliferative disorders, in immunocompromised individuals ( 3 ). EBV latency-II Ags such as Epstein-Barr nuclear Ag-1, latent membrane protein 1 (LMP1), and latent membrane protein 2A (LMP2A), are expressed in malignant cells ( 4 5 ). LMP2A mimics the activated B cell receptors (BCR) and inhibits normal signal transduction.…”