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.

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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.

The genetic transfer of T-cell receptors (TCRs) directed toward target antigens into T lymphocytes has been used to generate antitumor T cells efficiently without the need for the in vitro induction and expansion of T cells with cognate specificity. Alternatively, T cells have been gene-modified with a TCR-like antibody or chimeric antigen receptor (CAR). We show that immunization of HLA-A2 transgenic mice with tetramerized recombinant HLA-A2 incorporating HA-1 H minor histocompatibility antigen (mHag) peptides and β2-microglobulin (HA-1 H/HLA-A2) generate highly specific antibodies. One single-chain variable region moiety (scFv) antibody, #131, demonstrated high affinity (KD=14.9 nM) for the HA-1 H/HLA-A2 complex. Primary human T cells transduced with #131 scFV coupled to CD28 transmembrane and CD3ζ domains were stained with HA-1 H/HLA-A2 tetramers slightly more intensely than a cytotoxic T lymphocyte (CTL) clone specific for endogenously HLA-A2- and HA-1 H-positive cells. Although #131 scFv CAR-T cells required >100-fold higher antigen density to exert cytotoxicity compared with the cognate CTL clone, they could produce inflammatory cytokines against cells expressing HLA-A2 and HA-1 H transgenes. These data implicate that T cells with high-affinity antigen receptors reduce the ability to lyse targets with low-density peptide/MHC complexes (~100 per cell), while they could respond at cytokine production level.

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.