Candidate tumor-specific CD8+ T cell subsets identified in the malignant pleural effusion of advanced lung cancer patients by single-cell analysis.

Isolation of tumor-specific T cells and their antigen receptors (TCRs) from malignant pleural effusions (MPE) may facilitate the development of TCR-transduced adoptive cellular immunotherapy products for advanced lung cancer patients. However, the characteristics and markers of tumor-specific T-cells in MPE are largely undefined. To this end, to establish the phenotypes and antigen specificities of CD8+ T cells, we performed single-cell RNA and TCR sequencing of samples from three advanced lung cancer patients. Dimensionality reduction on a total of 4,983 CD8+ T cells revealed 10 clusters including naïve, memory, and exhausted phenotypes. We focused particularly on exhausted T cell clusters and tested their TCR reactivity against neoantigens predicted from autologous cancer cell lines. Four different TCRs specific for the same neoantigen and one orphan TCR specific for the autologous cell line were identified from one of the patients. Differential gene expression analysis in tumor-specific T cells relative to the other T cells identified CXCL13, as a candidate gene expressed by tumor-specific T cells. In addition to expressing CXCL13, tumor-specific T cells were present in a higher proportion of T cells co-expressing PDCD1(PD-1)/TNFRSF9(4-1BB). Furthermore, flow cytometric analyses in advanced lung cancer patients with MPE documented that those with high PD-1/4-1BB expression have a better prognosis in the subset of 57 adenocarcinoma patients (p = .039). These data suggest that PD-1/4-1BB co-expression might identify tumor-specific CD8+ T cells in MPE, which are associated with patients' prognosis. (233 words).

Single-cell sequencing on CD8+ TILs revealed the nature of exhausted T cells recognizing neoantigen and cancer/testis antigen in non-small cell lung cancer.

BACKGROUND: CD8+tumor infiltrating lymphocytes (TILs) are often observed in non-small cell lung cancers (NSCLC). However, the characteristics of CD8+ TILs, especially T-cell populations specific for tumor antigens, remain poorly understood. METHODS: High throughput single-cell RNA sequencing and single-cell T-cell receptor (TCR) sequencing were performed on CD8+ TILs from three surgically-resected lung cancer specimens. Dimensional reduction for clustering was performed using Uniform Manifold Approximation and Projection. CD8+ TIL TCR specific for the cancer/testis antigen KK-LC-1 and for predicted neoantigens were investigated. Differentially-expressed gene analysis, Gene Set Enrichment Analysis (GSEA) and single sample GSEA was performed to characterize antigen-specific T cells. RESULTS: A total of 6998 CD8+ T cells was analyzed, divided into 10 clusters according to their gene expression profile. An exhausted T-cell (exhausted T (Tex)) cluster characterized by the expression of ENTPD1 (CD39), TOX, PDCD1 (PD1), HAVCR2 (TIM3) and other genes, and by T-cell oligoclonality, was identified. The Tex TCR repertoire (Tex-TCRs) contained nine different TCR clonotypes recognizing five tumor antigens including a KK-LC-1 antigen and four neoantigens. By re-clustering the tumor antigen-specific T cells (n=140), it could be seen that the individual T-cell clonotypes were present on cells at different stages of differentiation and functional states even within the same Tex cluster. Stimulating these T cells with predicted cognate peptide indicated that TCR signal strength and subsequent T-cell proliferation and cytokine production was variable but always higher for neoantigens than KK-LC-1. CONCLUSIONS: Our approach focusing on T cells with an exhausted phenotype among CD8+ TILs may facilitate the identification of tumor antigens and clarify the nature of the antigen-specific T cells to specify the promising immunotherapeutic targets in patients with NSCLC.

Development of TCR-T cell therapy targeting mismatched HLA-DPB1 for relapsed leukemia after allogeneic transplantation.

Relapsed leukemia after allogeneic hematopoietic stem cell transplantation (allo-HSCT) remains a significant challenge, with the re-emergence of the primary disease being the most frequent cause of death. Human leukocyte antigen (HLA)-DPB1 mismatch occurs in approximately 70% of unrelated allo-HSCT cases, and targeting mismatched HLA-DPB1 is considered reasonable for treating relapsed leukemia following allo-HSCT if performed under proper conditions. In this study, we established several clones restricted to HLA-DPB1*02:01, -DPB1*04:02, and -DPB1*09:01 from three patients who underwent HLA-DPB1 mismatched allo-HSCT using donor-derived alloreactive T cells primed to mismatched HLA-DPB1 in the recipient's body after transplantation. A detailed analysis of the DPB1*09:01-restricted clone 2A9 showed reactivity against various leukemia cell lines and primary myeloid leukemia blasts, even with low HLA-DP expression. T cell receptor (TCR)-T cells derived from clone 2A9 retained the ability to trigger HLA-DPB1*09:01-restricted recognition and lysis of various leukemia cell lines in vitro. Our study demonstrated that the induction of mismatched HLA-DPB1 specific T cell clones from physiologically primed post-allo-HSCT alloreactive CD4+ T cells and the redirection of T cells with cloned TCR cDNA by gene transfer are feasible as techniques for future adoptive immunotherapy.

New evaluation of the tumor immune microenvironment of non-small cell lung cancer and its association with prognosis.

BACKGROUND: A better understanding of the tumor immune microenvironment (TIME) will facilitate the development of prognostic biomarkers and more effective therapeutic strategies in patients with lung cancer. However, little has been reported on the comprehensive evaluation of complex interactions among cancer cells, immune cells, and local immunosuppressive elements in the TIME. METHODS: Whole-exome sequencing and RNA sequencing were carried out on 113 lung cancers. We performed single sample gene set enrichment analysis on TIME-related gene sets to develop a new scoring system (TIME score), consisting of T-score (tumor proliferation), I-score (antitumor immunity) and S-score (immunosuppression). Lung cancers were classified according to a combination of high or low T-score, I-score, and S-scores (eight groups; G1-8). Clinical and genomic features, and immune landscape were investigated among eight groups. The external data sets of 990 lung cancers from The Cancer Genome Atlas and 76 melanomas treated with immune checkpoint inhibitors (ICI) were utilized to evaluate TIME scoring and explore prognostic and predictive accuracy. RESULTS: The representative histological type including adenocarcinoma and squamous cell carcinoma, and driver mutations such as epidermal growth factor receptor and TP53 mutations were different according to the T-score. The numbers of somatic mutations and predicted neoantigens were higher in Thi (G5-8) than Tlo (G1-4) tumors. Immune selection pressure against neoantigen expression occurred only in Thi and was dampened in Thi/Ilo (G5-6), possibly due to a reduced number of T cells with a high proportion of tumor specific but exhausted cells. Thi/Ilo/Shi (G5) displayed the lowest immune responses by additional immune suppressive mechanisms. The T-score, I-score and S-scores were independent prognostic factors, with survival curves well separated into eight groups with G5 displaying the worst overall survival, while the opposite group Tlo/Ihi/Slo (G4) had the best prognosis. Several oncogenic signaling pathways influenced on T-score and I-scores but not S-score, and PI3K pathway alteration correlated with poor prognosis in accordance with higher T-score and lower I-score. Moreover, the TIME score predicted the efficacy of ICI in patients with melanoma. CONCLUSION: The TIME score capturing complex interactions among tumor proliferation, antitumor immunity and immunosuppression could be useful for prognostic predictions or selection of treatment strategies in patients with lung cancer.

Development and Accuracy Evaluation of Augmented Reality-based Patient Positioning System in Radiotherapy: A Phantom Study.

BACKGROUND/AIM: To develop and evaluate the accuracy of augmented reality (AR)-based patient positioning systems in radiotherapy. MATERIALS AND METHODS: AR head-mounted displays (AR-HMDs), which virtually superimpose a three-dimensional (3D) image generated by the digital imaging and communications in medicine (DICOM) data, have been developed. The AR-based positioning feasibility was evaluated. Then, the setup errors of three translational axes directions and rotation angles between the AR and the conventional laser-based positioning were compared. RESULTS: The AR-based pelvic phantom positioning was feasible. The setup errors of AR-based positioning were comparable to laser-based positioning in all translational axis directions and rotation angles. The time necessary for AR-based positioning was significantly longer than that for laser-based positioning (171.0 s vs. 47.5 s, p<0.001). CONCLUSION: AR-based positioning for radiotherapy was feasible, and showed comparable positioning errors to those of conventional line-based positioning; however, a markedly longer setup time was necessary.

[Identification of neoantigens and development of antigen-specific immunotherapy].

Cancer cells harboring somatic mutations give rise to neoantigens, which are immunologically foreign in nature to be distinguished from itself, showing high immunogenicity and, thus, induce specific T-cell responses against cancer. Therefore, neoantigens are expected to be promising targets for anti-cancer immunotherapy. The general methods used to identify candidate neoantigens are as follows: (1) non-synonymous mutations are identified by whole exome and RNA sequencing; (2) neoantigens from the mutations are predicted based on in silico MHC ligand prediction algorithm; (3) specific T-cell responses toward the candidate neoantigens are verified using tumor infiltrating T cells or peripheral blood mononuclear cells. In hematological malignancy, several neoantigens have been identified as an important treatment target. In contrast with solid malignancies, the occurrence of frameshift mutations and fusion genes producing neoantigens are high. A shared neoantigen derived from frameshift mutation of nucleophosmin I, which is often observed in acute myeloid leukemia, was reported to induce specific immune responses in vitro and in vivo. We should examine neoantigens as possible target of novel immunotherapy despite several issues to be addressed for clinical application.

[Neoantigens Are Critical Targets in Naturally and Therapeutically Induced Immune Responses to Cancer].

T cells are critical effector immune cells, and mutation-derived neoantigens are critical tumor-specific antigens in natural immune responses to cancer(cancer immunosurveillance). However, the underlying mechanisms are poorly understood, particularly in the human clinical setting, such as how many tumor antigens are related to cancer elimination and whether immunodominance of antigens exist in humans. Furthermore, it is unclear whether specific T cells recognizing neoantigens can control cancer for a long time in an equilibrium state. Cancer immunotherapy including immune checkpoint inhibitors (ICIs)such as PD-1 and CTLA-4 blockades has attracted much attention for cancer treatment, but they remain effective only in a minority of patients. However, the efficacy of ICIs, which is characterized by long term durable responses has suggested that host immunity, if re-activated, can eliminate cancer cells or maintain cancer for a long time(therapeutically induced cancer immune elimination or equilibrium). Recent reports show that the loss of neoantigens has occurred at the DNA and RNA level as results of both naturally and therapeutically induced tumor-specific immune responses, leading to tumor immune escape(re-escape). Here we focus on the role of neoantigens in naturally and therapeutically induced immunoediting revealed by cancer immunogenomics approaches utilizing clinical samples to develop effective neoantigen-based cancer immunotherapies.

Frequent structural variations involving programmed death ligands in Epstein-Barr virus-associated lymphomas.

Viral infection induces potent cellular immunity and activated intracellular signaling, which may dictate the driver events involved in immune escape and clonal selection of virus-associated cancers, including Epstein-Barr virus (EBV)-positive lymphomas. Here, we thoroughly interrogated PD-L1/PD-L2-involving somatic aberrations in 384 samples from various lymphoma subtypes using high-throughput sequencing, particularly focusing on virus-associated lymphomas. A high frequency of PD-L1/PD-L2-involving genetic aberrations was observed in EBV-positive lymphomas [33 (22%) of 148 cases], including extranodal NK/T-cell lymphoma (ENKTL, 23%), aggressive NK-cell leukemia (57%), systemic EBV-positive T-cell lymphoproliferative disorder (17%) as well as EBV-positive diffuse large B-cell lymphoma (DLBCL, 19%) and peripheral T-cell lymphoma-not otherwise specified (15%). Predominantly causing a truncation of the 3'-untranslated region, these alterations represented the most prevalent somatic lesions in ENKTL. By contrast, the frequency was much lower in EBV-negative lymphomas regardless of histology type [12 (5%) of 236 cases]. Besides PD-L1/PD-L2 alterations, EBV-positive DLBCL exhibited a genetic profile distinct from EBV-negative one, characterized by frequent TET2 and DNMT3A mutations and the paucity of CD79B, MYD88, CDKN2A, and FAS alterations. Our findings illustrate unique genetic features of EBV-associated lymphomas, also suggesting a potential role of detecting PD-L1/PD-L2-involving lesions for these lymphomas to be effectively targeted by immune checkpoint blockade.

Improving TCR affinity on 293T cells.

This study presents an efficient method to improve TCR affinity, comprising 1) CDR-directed saturation mutation of TCR cDNA, 2) transient TCR display on CD3-expressing HEK293T (CD3-293T) cells by simple plasmid transfection, 3) staining with HLA-tetramers, and 4) multi-round sorting of cells with CD8-independent tetramer binding on a flow cytometer. Using these procedures, we successfully identified mutant TCRs with enhanced binding from an HLA-A*24:02-restricted, human telomerase reverse transcriptase (hTERT)-specific TCR. Two such clones, 2A7A and 2D162, harboring mutations in CDR1 and CDR2 of TCRß, respectively, were isolated with both showing sequential four amino acid substitutions. When expressed on CD3-293T cells along with wild-type TCRα, the TCR molecules of these mutants as well as their combinatory mutation, bound to HLA-A24/hTERT-tetramers more strongly than the wild-type TCRs, without binding to control tetramers. Besides, in order to facilitate a functional study of TCR, we established an artificial T cell line, designated as CD8I-J2, which expresses a human CD8 and IFN-γ producing cassette by modifying Jurkat-derived J.RT3-T3.5 cells. CD8I-J2 cells expressing wild-type or affinity-enhanced hTERT-specific TCRs were analyzed for their recognition of serially diluted cognate peptide on HLA-A*24:02-transduced T2 cells. CD8I-J2 cells expressing each mutant TCR recognized the hTERT peptide at lower concentrations than wild-type TCR. The hierarchy of peptide recognition is concordant with tetramer binding on CD3-293T cells and none of these mutant TCRs were cross-reactive with irrelevant peptides reported to be present on HLA-A*24:02 molecules as far as tested. These methods might thus be useful for obtaining high affinity mutants from other TCRs of interest.

Changes in prolactin receptor homodimer availability may cause late feathering in chickens.

Chicken early (EF) and late feathering (LF) are sex-linked phenotypes conferred by wild-type k+ and dominant K alleles on chromosome Z, respectively. Besides prolactin (PRL) receptor (PRLR) and sperm flagellar 2 (SPEF2) genes, the K allele contains a fusion gene in which partially duplicated PRLR (dPRLR) and SPEF2 (dSPEF2) genes are linked in a tail-to-tail manner. The causative dPRLR gene encodes a C-terminal truncated receptor. LF chickens have short or no primaries at hatching; however, their feather growth rate is higher than that of EF chickens. This study aimed to elucidate the molecular basis of the K allele's biphasic effect on feather development. By 3'RACE and RT-PCR analyses, we demonstrated that dSPEF2 gene transcription occurred beyond all coding exons of the dPRLR gene on the opposite strand and that dPRLR mRNA was less abundant than PRLR mRNA. In addition, a 5'UTR splice variant (SPV) of PRL receptor mRNAs was increased in LF chickens. In vitro expression analysis of 5'UTR linked to the luciferase reporter gene revealed higher translation efficiency of SPV. RT-qPCR showed that the dPRLR mRNA level was higher in embryos; conversely, SPV was higher in hatched chickens, as was dSPEF2 mRNA. These findings suggest that the K allele inhibits feather development at the fetal stage by expressing dPRLR to attenuate PRLR function and promotes feather growth after hatching by increasing PRLR through dSPEF2 mRNA expression. Increased SPV may cause greater feather growth than that in EF chickens by increasing the availability of PRLR homodimers and enhancing PRL signaling.

Bile acid-regulated peroxisome proliferator-activated receptor-α (PPARα) activity underlies circadian expression of intestinal peptide absorption transporter PepT1/Slc15a1.

Digested proteins are mainly absorbed as small peptides composed of two or three amino acids. The intestinal absorption of small peptides is mediated via only one transport system: the proton-coupled peptide transporter-1 (PepT1) encoded from the soluble carrier protein Slc15a1. In mammals, intestinal expression of PepT1/Slc15a1 oscillates during the daily feeding cycle. Although the oscillation in the intestinal expression of PepT1/Slc15a1 is suggested to be controlled by molecular components of circadian clock, we demonstrated here that bile acids regulated the oscillation of PepT1/Slc15a1 expression through modulating the activity of peroxisome proliferator-activated receptor α (PPARα). Nocturnally active mice mainly consumed their food during the dark phase. PPARα activated the intestinal expression of Slc15a1 mRNA during the light period, and protein levels of PepT1 peaked before the start of the dark phase. After food intake, bile acids accumulated in intestinal epithelial cells. Intestinal accumulated bile acids interfered with recruitment of co-transcriptional activator CREB-binding protein/p300 on the promoter region of Slc15a1 gene, thereby suppressing PPARα-mediated transactivation of Slc15a1. The time-dependent suppression of PPARα-mediated transactivation by bile acids caused an oscillation in the intestinal expression of PepT1/Slc15a1 during the daily feeding cycle that led to circadian changes in the intestinal absorption of small peptides. These findings suggest a molecular clock-independent mechanism by which bile acid-regulated PPARα activity governs the circadian expression of intestinal peptide transporter.

Neutrophil gelatinase-associated lipocalin levels associated with cardiovascular disease in chronic kidney disease patients.

BACKGROUND: Elevated levels of neutrophil gelatinase-associated lipocalin (NGAL) have been reported in patients with cardiovascular disease (CVD), heart failure, and stroke. We assessed the relationships between serum levels of NGAL and the prevalence of CVD, and clarified the prognostic usefulness of systemic NGAL levels in hemodialysis (HD) patients. METHODS: Eighty-eight HD patients were followed up for 1 year. Logistic regression analyses were used to investigate the relationship between de novo CVD status and NGAL levels as well as other risk factors. RESULTS: During follow-up, CVD events occurred in 20 patients. Initial serum levels of NGAL and brain natriuretic peptide of HD patients with de novo CVD were significantly higher than those of HD patients without de novo CVD. Multivariate logistic regression analyses showed that initial serum levels of NGAL were independent risk factors for de novo CVD in HD patients. When patients were classified on the basis of NGAL quartiles, multiple logistic regression analyses demonstrated that the highest quartile of NGAL level showed an increased odds ratio for the prevalence of CVD. CONCLUSION: These findings suggest that NGAL levels can be used to detect the prevalence of CVD in HD patients with or without diabetes.

An HLA-modified ovarian cancer cell line induced CTL responses specific to an epitope derived from claudin-1 presented by HLA-A*24:02 molecules.

In an attempt to induce cytotoxic T lymphocytes (CTLs) that react to ovarian cancer cells, we isolated a CTL clone that specifically recognizes claudin-1 in an HLA-A*24:02-restricted manner. Naïve CD8(+) T lymphocytes were obtained from a healthy adult donor and stimulated twice in vitro with HLA-modified TOV21G cells that were originally derived from an ovarian clear-cell carcinoma line. The TOV21G modification involved RNAi-mediated gene silencing of intrinsic HLA molecules and lentiviral transduction of a synonymously mutated HLA-A*24:02. Then, cDNA library construction using mRNA extracted from the parental TOV21G cells and subsequent expression cloning were conducted. These experiments revealed that a CTL clone obtained from the bulk culture recognized a minimal epitope peptide RYEFGQALF, which was derived from an autoantigen claudin-1 presented by HLA-A*24:02 molecules. This clone exhibited cytolytic activities against three ovarian cancer cell lines and normal bronchial epithelial cells in an HLA-A*24:02-restricted manner. Our data indicate that HLA-modified cancer cells can be used as an artificial antigen-presenting cell to generate antigen-specific CTLs in a manner restricted by an HLA allele of interest.

Autophagy creates a CTL epitope that mimics tumor-associated antigens.

The detailed mechanisms responsible for processing tumor-associated antigens and presenting them to CTLs remain to be fully elucidated. In this study, we demonstrate a unique CTL epitope generated from the ubiquitous protein puromycin-sensitive aminopeptidase, which is presented via HLA-A24 on leukemic and pancreatic cancer cells but not on normal fibroblasts or EBV-transformed B lymphoblastoid cells. The generation of this epitope requires proteasomal digestion and transportation from the endoplasmic reticulum to the Golgi apparatus and is sensitive to chloroquine-induced inhibition of acidification inside the endosome/lysosome. Epitope liberation depends on constitutively active autophagy, as confirmed with immunocytochemistry for the autophagosome marker LC3 as well as RNA interference targeting two different autophagy-related genes. Therefore, ubiquitously expressed proteins may be sources of specific tumor-associated antigens when processed through a unique mechanism involving autophagy.

Zoledronate sensitizes neuroblastoma-derived tumor-initiating cells to cytolysis mediated by human γδ T cells.

Neuroblastoma is the most common extracranial solid tumor in children that is refractory to intensive multimodal therapy. In particular, tumor-initiating cells (TICs) derived from neuroblastoma are believed responsible for tumor formation and resistance to the conventional therapy; an optimal strategy therefore should target this population. Technically, TICs can be enriched from neuroblastoma-derived spheres when the tumor cells are cultured in a serum-free medium supplemented with certain growth factors. Recently, a line of evidence has suggested antitumor potential of Vγ9Vδ2 T cells (γδ T cells), a T-cell population that recognizes and kills target cells independent of surface HLA expressions. Furthermore, a mevalonate pathway inhibitor, zoledronate, has been reported to enhance cytolytic activity of γδ T cells. On the basis of these findings, we hypothesized that zoledronate would sensitize neuroblastoma TICs to γδ T-cell-mediated cytolysis and promote therapeutic efficacy against neuroblastoma. In the current study, we show that zoledronate efficiently sensitizes both neuroblastoma-derived adherent cells and sphere-forming cells to γδ T-cell-mediated cytolysis. Subsequently, in vitro colony formation inhibition assay and in vivo animal studies reveal that the presence of γδ T cells decelerates outgrowth of neuroblastoma TICs. We finally show that addition of interleukin-15 and/or interleukin-18 in culture enhances the cytolytic activity of γδ T cells. On the basis of these data, we conclude that ex vivo expanded γδ T cells are a promising tool for antineuroblastoma immunotherapy with options for further improvement.

Mismatched human leukocyte antigen class II-restricted CD8⁺ cytotoxic T cells may mediate selective graft-versus-leukemia effects following allogeneic hematopoietic cell transplantation.

Partial human leukocyte antigen (HLA)-mismatched hematopoietic stem cell transplantation (HSCT) is often performed when an HLA-matched donor is not available. In these cases, CD8(+) or CD4(+) T cell responses are induced depending on the mismatched HLA class I or II allele(s). Herein, we report on an HLA-DRB1*08:03-restricted CD8(+) CTL clone, named CTL-1H8, isolated from a patient following an HLA-DR-mismatched HSCT from his brother. Lysis of a patient Epstein-Barr virus-transformed B cell line (B-LCL) by CTL-1H8 was inhibited after the addition of blocking antibodies against HLA-DR and CD8, whereas antibodies against pan-HLA class I or CD4 had no effect. The 1H8-CTL clone did not lyse the recipient dermal fibroblasts whose HLA-DRB1*08:03 expression was upregulated after 1 week cytokine treatment. Engraftment of HLA-DRB1*08:03-positive primary leukemic stem cells in non-obese diabetic/severe combined immunodeficient/γc-null (NOG) mice was completely inhibited by the in vitro preincubation of cells with CTL-1H8, suggesting that HLA-DRB1*08:03 is expressed on leukemic stem cells. Finally, analysis of the precursor frequency of CD8(+) CTL specific for recipient antigens in post-HSCT peripheral blood T cells revealed a significant fraction of the total donor CTL responses towards the individual mismatched HLA-DR antigen in two patients. These findings underscore unexpectedly significant CD8 T cell responses in the context of HLA class II.

Quantitative analysis of Epstein-Barr virus (EBV)-related gene expression in patients with chronic active EBV infection.

Chronic active Epstein-Barr virus (CAEBV) infection is a systemic Epstein-Barr virus (EBV)-positive lymphoproliferative disorder characterized by persistent or recurrent infectious mononucleosis-like symptoms in patients with no known immunodeficiency. The detailed pathogenesis of the disease is unknown and no standard treatment regimen has been developed. EBV gene expression was analysed in peripheral blood samples collected from 24 patients with CAEBV infection. The expression levels of six latent and two lytic EBV genes were quantified by real-time RT-PCR. EBV-encoded small RNA 1 and BamHI-A rightward transcripts were abundantly detected in all patients, and latent membrane protein (LMP) 2 was observed in most patients. EBV nuclear antigen (EBNA) 1 and LMP1 were detected less frequently and were expressed at lower levels. EBNA2 and the two lytic genes were not detected in any of the patients. The pattern of latent gene expression was determined to be latency type II. EBNA1 was detected more frequently and at higher levels in the clinically active patients. Quantifying EBV gene expression is useful in clarifying the pathogenesis of CAEBV infection and may provide information regarding a patient's disease prognosis, as well as possible therapeutic interventions.

Epstein-Barr virus nuclear antigen 1-specific CD4+ T cells directly kill Epstein-Barr virus-carrying natural killer and T cells.

Epstein-Barr virus (EBV) nuclear antigen (EBNA)1 is expressed in every EBV-infected cell, regardless of the state of EBV infection. Although EBNA1 is thought to be a promising antigen for immunotherapy of all EBV-associated malignancies, it is less clear whether EBNA1-specific CD4(+) T cells can act as direct effectors. Herein, we investigated the ability of CD4(+) T-cell clones induced with overlapping peptides covering the C-terminal region of EBNA1, and identified minimal epitopes and their restricted major histocompatibility complex class II molecules. Of these, a novel epitope, EYHQEGGPD, was found to be presented by DRB1*0401, 0403 and 0406. Five CD4(+) T-cell clones recognized endogenously processed and presented antigens on EBV-transformed lymphoblastoid cell lines (LCL) and one example proved capable of killing EBV-carrying natural killer (NK) and T-cell lines derived from patients with chronic active EBV infection (CAEBV). Identification of minimal epitopes facilitates design of peptide-based vaccines and our data suggest that EBNA1-specific CD4(+) T cells may play roles as direct effectors for immunotherapy targeting EBV-carrying NK and T-cell malignancies.