EBV-transformed Lymphoblastoid Cell Lines Research Articles

EBV T-cell immunotherapy generated by peptide selection has enhanced effector functionality compared to LCL stimulation.

Adoptive immunotherapy with Epstein-Barr virus (EBV)-specific T cells is an effective treatment for relapsed or refractory EBV-induced post-transplant lymphoproliferative disorders (PTLD) with overall survival rates of up to 69%. EBV-specific T cells have been conventionally made by repeated stimulation with EBV-transformed lymphoblastoid cell lines (LCL), which act as antigen-presenting cells. However, this process is expensive, takes many months, and has practical risks associated with live virus. We have developed a peptide-based, virus-free, serum-free closed system to manufacture a bank of virus-specific T cells (VST) for clinical use. We compared these with standard LCL-derived VST using comprehensive characterization and potency assays to determine differences that might influence clinical benefits. Multi-parameter flow cytometry revealed that peptide-derived VST had an expanded central memory population and less exhaustion marker expression than LCL-derived VST. A quantitative HLA-matched allogeneic cytotoxicity assay demonstrated similar specific killing of EBV-infected targets, though peptide-derived EBV T cells had a significantly higher expression of antiviral cytokines and degranulation markers after antigen recall. High-throughput T cell receptor-beta (TCRβ) sequencing demonstrated oligoclonal repertoires, with more matches to known EBV-binding complementary determining region 3 (CDR3) sequences in peptide-derived EBV T cells. Peptide-derived products showed broader and enhanced specificities to EBV nuclear antigens (EBNAs) in both CD8 and CD4 compartments, which may improve the targeting of highly expressed latency antigens in PTLD. Importantly, peptide-based isolation and expansion allows rapid manufacture and significantly increased product yield over conventional LCL-based approaches.

CD56bright NK cell expansion correlated with EBV reactivation control post allogeneic hematopoietic stem cell transplantation.

Natural killer (NK) cells are equipped with anti-Epstein-Barr virus (EBV) function, however, whether EBV infection will affect NK cells reconstitution after allogeneic hematopoietic stem cell transplantation (allo-HSCT) remains unclear. To identify the characteristics of NK cells, we prospectively enrolled 11 patients who occurred EBV reactivation post allo-HSCT and 11 patients without EBV infection as control. We found that that EBV infection induced the expansion of CD56bright and NKG2A+KIR- NK subsets,and decreased the cytotoxicity function of NK cells. The frequency of NKG2A+KIR- NK cells were higher in patients who progressed into post-transplant lymphoproliferative disorder (PTLD) than EBV viremia patients, which also correlated with decreased proliferation and cytotoxic function. By screening the activation receptors of NK cells, we found the DNAM-1+CD56bright NK cells is significantly increased after EBV stimulation, further we demonstrated that DNAM-1 is essential for EBV induced NK cells activation as the cytokine release against EBV-transformed lymphoblastoid cell lines(EBV-LCLs) of CD56bright NK cells were significantly decreased after DNAM-1 blockade. NK cells infusion suppressed the progression of EBV-related tumor mice model. A prospective cohort indicated that old donor age was an independent risk factor for EBV infection. Rapid CD56bri expansion and high expression of DNAM-1 on CD56bri NK cells in response to EBV reactivation correlated with rapid EBV clearance post allo-HSCT in patients with younger donors. In summary, our data showed that high expression of DNAM-1 receptors on NK cell may participate protective CD56bri NK cells response to EBV infection after allo-HSCT.

LMP1 and EBNA2 constitute a minimal set of EBV genes for transformation of human B cells.

Epstein-Barr virus (EBV) infection in humans is associated with a wide range of diseases including malignancies of different origins, most prominently B cells. Several EBV latent genes are thought to act together in B cell immortalization, but a minimal set of EBV genes sufficient for transformation remains to be identified. Here, we addressed this question by transducing human peripheral B cells from EBV-negative donors with retrovirus expressing the latent EBV genes encoding Latent Membrane Protein (LMP) 1 and 2A and Epstein-Barr Nuclear Antigen (EBNA) 2. LMP1 together with EBNA2, but not LMP1 alone or in combination with LMP2A was able to transform human primary B cells. LMP1/EBNA2-immortalized cell lines shared surface markers with EBV-transformed lymphoblastoid cell lines (LCLs). They showed sustained growth for more than 60 days, albeit at a lower growth rate than EBV-transformed LCLs. LMP1/EBNA2-immortalized cell lines generated tumors when transplanted subcutaneously into severely immunodeficient NOG mice. Our results identify a minimal set of EBV proteins sufficient for B cell transformation.

Spontaneous EBV-Reactivation during B Cell Differentiation as a Model for Polymorphic EBV-Driven Lymphoproliferation.

Epstein-Barr virus (EBV)-driven B cell neoplasms arise from the reactivation of latently infected B cells. In a subset of patients, EBV was seen to drive a polymorphous lymphoproliferative disorder (LPD) in which B cell differentiation was retained. In this work, spontaneous EBV reactivation following B cell mitogen stimulation was shown to provide a potential model of polymorphic EBV-driven LPD. Here, we developed an in vitro model of plasma cell (PC) differentiation from peripheral blood memory B cells. To assess the frequency and phenotypes of EBV-associated populations derived during differentiation, we analysed eight differentiations during the PC stage with a targeted single-cell gene expression panel. We identified subpopulations of EBV-gene expressing cells with PC and/or B cell expression features in differentiations from all tested donors. EBV-associated cells varied in frequency, ranging from 3-28% of cells. Most EBV-associated cells expressed PC genes such as XBP1 or MZB1, and in all samples these included a quiescent PC fraction that lacked cell a cycle gene expression. With increasing EBV-associated cells, populations with B cell features became prominent, co-expressing a germinal centre (GC) and activating B cell gene patterns. The presence of highly proliferative EBV-associated cells was linked to retained MS4A1/CD20 expression and IGHM and IGHD co-expression, while IGHM class-switched cells were enriched in quiescent PC fractions. Thus, patterns of gene expression in primary EBV reactivation were shown to include features related to GC B cells, which was also observed in EBV-transformed lymphoblastoid cell lines. This suggests a particular association between spontaneously developing EBV-expansions and IgM+ IgD+ non-switched B cells.

The Epstein-Barr Virus Enhancer Interaction Landscapes in Virus-Associated Cancer Cell Lines.

Epstein-Barr virus (EBV) persists in human cells as episomes. EBV episomes are chromatinized and their 3D conformation varies greatly in cells expressing different latency genes. We used HiChIP, an assay which combines genome-wide chromatin conformation capture followed by deep sequencing (Hi-C) and chromatin immunoprecipitation (ChIP), to interrogate the EBV episome 3D conformation in different cancer cell lines. In an EBV-transformed lymphoblastoid cell line (LCL) GM12878 expressing type III EBV latency genes, abundant genomic interactions were identified by H3K27ac HiChIP. A strong enhancer was located near the BILF2 gene and looped to multiple genes around BALFs loci. Perturbation of the BILF2 enhancer by CRISPR interference (CRISPRi) and CRISPR activation (CRISPRa) altered the expression of BILF2 enhancer-linked genes, including BARF0 and BALF2, suggesting that this enhancer regulates the expression of linked genes. H3K27ac ChIP followed by deep sequencing (ChIP-seq) identified several strong EBV enhancers in T/NK (natural killer) lymphoma cells that express type II EBV latency genes. Extensive intragenomic interactions were also found which linked enhancers to target genes. A strong enhancer at BILF2 also looped to the BALF loci. CRISPRi also validated the functional connection between BILF2 enhancer and BARF1 gene. In contrast, H3K27ac HiChIP found significantly fewer intragenomic interactions in type I EBV latency gene-expressing primary effusion lymphoma (PEL) cell lines. These data provided new insight into the regulation of EBV latency gene expression in different EBV-associated tumors. IMPORTANCE EBV is the first human DNA tumor virus identified, discovered over 50 years ago. EBV causes ~200,000 cases of various cancers each year. EBV-encoded oncogenes, noncoding RNAs, and microRNAs (miRNAs) can promote cell growth and survival and suppress senescence. Regulation of EBV gene expression is very complex. The viral C promoter regulates the expression of all EBV nuclear antigens (EBNAs), some of which are very far away from the C promoter. Another way by which the virus activates remote gene expression is through DNA looping. In this study, we describe the viral genome looping patterns in various EBV-associated cancer cell lines and identify important EBV enhancers in these cells. This study also identified novel opportunities to perturb and eventually control EBV gene expression in these cancer cells.

Chagas Disease Megaesophagus Patients Carrying Variant MRPS18B P260A Display Nitro-Oxidative Stress and Mitochondrial Dysfunction in Response to IFN-γ Stimulus.

Chagas disease (CD), caused by the protozoan parasite Trypanosoma cruzi, affects 8 million people, and around 1/3 develop chronic cardiac (CCC) or digestive disease (megaesophagus/megacolon), while the majority remain asymptomatic, in the indeterminate form of Chagas disease (ASY). Most CCC cases in families with multiple Chagas disease patients carry damaging mutations in mitochondrial genes. We searched for exonic mutations associated to chagasic megaesophagus (CME) in genes essential to mitochondrial processes. We performed whole exome sequencing of 13 CME and 45 ASY patients. We found the damaging variant MRPS18B 688C > G P230A, in five out of the 13 CME patients (one of them being homozygous; 38.4%), while the variant appeared in one out of 45 ASY patients (2.2%). We analyzed the interferon (IFN)-γ-induced nitro-oxidative stress and mitochondrial function of EBV-transformed lymphoblastoid cell lines. We found the CME carriers of the mutation displayed increased levels of nitrite and nitrated proteins; in addition, the homozygous (G/G) CME patient also showed increased mitochondrial superoxide and reduced levels of ATP production. The results suggest that pathogenic mitochondrial mutations may contribute to cytokine-induced nitro-oxidative stress and mitochondrial dysfunction. We hypothesize that, in mutation carriers, IFN-γ produced in the esophageal myenteric plexus might cause nitro-oxidative stress and mitochondrial dysfunction in neurons, contributing to megaesophagus.

Pathogenic variants detected by RNA sequencing in Cornelia de Lange syndrome

Recent studies suggest that transcript isoforms significantly overlap (approximately 60%) between brain tissue and Epstein–Barr virus-transformed lymphoblastoid cell lines (LCLs). Interestingly, 14 cohesion-related genes with variants that cause Cornelia de Lange Syndrome (CdLS) are highly expressed in the brain and LCLs. In this context, we first performed RNA sequencing of LCLs from 22 solved (with pathogenic variants) and 19 unsolved (with no confirmed variants) CdLS cases. Next, an RNA sequencing pipeline was developed using solved cases with two different methods: short variant analysis (for single-nucleotide and indel variants) and aberrant splicing detection analysis. Then, 19 unsolved cases were subsequently applied to our pipeline, and four pathogenic variants in NIPBL (one inframe deletion and three intronic variants) were newly identified. Two of three intronic variants were located at Alu elements in deep-intronic regions, creating cryptic exons. RNA sequencing with LCLs was useful for identifying hidden variants in exome-negative cases.

BRCA1 Variant Assessment Using a Simple Analytic Assay.

We previously developed a biological assay to accurately predict BRCA1 (BRCA1 DNA repair associated) mutation status, based on gene expression profiles of Epstein-Barr virus-transformed lymphoblastoid cell lines. The original work was done using whole genome expression microarrays, and nearest shrunken centroids analysis. While these approaches are appropriate for model building, they are difficult to implement clinically, where more targeted testing and analysis are required for time and cost savings. Here, we describe adaptation of the original predictor to use the NanoString nCounter platform for testing, with analysis based on the k-top scoring pairs (k-TSP) method. Assessing gene expression using the nCounter platform on a set of lymphoblastoid cell lines yielded 93.8% agreement with the microarray-derived data, and 87.5% overall correct classification of BRCA1 carriers and controls. Using the original gene expression microarray data used to develop our predictor with nearest shrunken centroids, we rebuilt a classifier based on the k-TSP method. This classifier relies on the relative expression of 10 pairs of genes, compared to the original 43 identified by nearest shrunken centroids (NSC), and was 96.2% concordant with the original training set prediction, with a 94.3% overall correct classification of BRCA1 carriers and controls. The k-TSP classifier was shown to accurately predict BRCA1 status using data generated on the nCounter platform and is feasible for initiating a clinical validation.

Non-Random Pattern of Integration for Epstein-Barr Virus with Preference for Gene-Poor Genomic Chromosomal Regions into the Genome of Burkitt Lymphoma Cell Lines

Background: Epstein-Barr virus (EBV) is an oncogenic virus found in about 95% of endemic Burkitt lymphoma (BL) cases. In latently infected cells, EBV DNA is mostly maintained in episomal form, but it can also be integrated into the host genome, or both forms can coexist in the infected cells. Methods: In this study, we mapped the chromosomal integration sites of EBV (EBV-IS) into the genome of 21 EBV+ BL cell lines (BL-CL) using metaphase fluorescence in situ hybridization (FISH). The data were used to investigate the EBV-IS distribution pattern in BL-CL, its relation to the genome instability, and to assess its association to common fragile sites and episomes. Results: We detected a total of 459 EBV-IS integrated into multiple genome localizations with a preference for gene-poor chromosomes. We did not observe any preferential affinity of EBV to integrate into common and rare fragile sites or enrichment of EBV-IS at the chromosomal breakpoints of the BL-CL analyzed here, as other DNA viruses do. Conclusions: We identified a non-random integration pattern into 13 cytobands, of which eight overlap with the EBV-IS in EBV-transformed lymphoblastoid cell lines and with a preference for gene- and CpGs-poor G-positive cytobands. Moreover, it has been demonstrated that the episomal form of EBV interacts in a non-random manner with gene-poor and AT-rich regions in EBV+ cell lines, which may explain the observed affinity for G-positive cytobands in the EBV integration process. Our results provide new insights into the patterns of EBV integration in BL-CL at the chromosomal level, revealing an unexpected connection between the episomal and integrated forms of EBV.

Tc Buster Transposon Engineered CLL-1 CAR-NK Cells Efficiently Target Acute Myeloid Leukemia

Introduction:DNA transposons are efficient, integrating, non-viral vector systems which have been applied to clinical scale CAR-T cell production, aiming to reduce cost and deliver larger genetic cargos relative to viral transduction. CAR-NK cells are a promising cell therapy platform, with a potential for ‘off-the-shelf’, allogeneic application. Inefficient delivery of plasmid DNA and substantial associated toxicity have limited the utility of DNA transposon systems in primary NK cell engineering. Here we describe an efficient process for the production of transposon engineered primary CAR-NK cells, and the in vitro activity of a CAR-NK cell product targeting human C-type lectin-like molecule-1 (CLL-1/ C-type lectin domain family 12 member A, CLEC12A) for acute myeloid leukemia (AML) produced using the Tc Buster (TcB) transposon system. Since CLL-1 is frequently expressed by leukemic stem cells (LSC) but absent on hematopoietic stem cells, it represents a rational antigenic target to enhance the innate activity of allogeneic NK cell therapies in AML.Methods:NK cells were isolated from 30ml of healthy donor peripheral blood by negative immunomagnetic selection (NK Cell Isolation Kit, Miltenyi Biotec), then activated and expanded using a good manufacturing practice (GMP) grade, 100 Gy irradiated, Epstein Barr Virus-transformed lymphoblastoid cell line (EBV-LCL) in the presence of IL-2 and IL-21. On day 4, NK cells were electroporated with a nanoplasmid TcB transposon carrying a second generation CLL-1 CAR (CD28/CD3ζ or 41BB/CD3ζ) and hyperactive TcB transposase mRNA (Maxcyte ATx). Following transposition, the NK cells were stimulated with the feeder line for a second time. Purified CAR-NK cell populations were produced by immunomagnetic selection of CAR expressing cells using anti-biotin beads (Miltenyi Biotec) and biotinylated CLL-1 protein (ACRO Biosystems), which was also used to detect CAR expression by flow cytometry. For CRISPR/Cas9 gene knockout (KO), pooled sgRNAs targeting 3 sites within the target gene were complexed with Cas9 protein prior to co-electroporation with the TcB payload. CLL-1 CAR-NK cell function was evaluated in co-culture with AML cell lines or biobanked AML patient samples and analyzed by flow cytometry.Results:This approach (Fig 1A) produced efficient transposition with retained clinical-scale expansion capacity of primary CAR-NK cells with mean CAR expression of 37% (n=3, range 26-46%) at day 21, without selection (Fig 1B). By extrapolation, a mean 4,275-fold expansion was observed by day 21, increasing to 14,023-fold by day 25, sufficient to support many clinical doses of 1x10 7 cells/Kg (Fig 1C). Mean NK cell purity of the final product was 98%. A further increase in the proportion of CAR-NK cells was achieved by immunomagnetic selection, with mean CAR expression increasing to 89.5% (range 84-97%, n=3). A further feeder cell stimulation of these CLL-1 CAR selected cells produced a similar expansion trajectory, 3 days delayed relative to unselected cells (Fig 1C). CLL-1 CAR-NK cell function was confirmed by an enhanced ability to target CLL-1 positive AML cell lines and primary AML blasts relative to control electroporated NK cells (Fig 1 D,E). Increased elimination of a primary AML population enriched for LSC (CD34+, CD38-, CLL-1+) by CLL-1 CAR-NK cells was confirmed (mean 96% vs. 32%, p=0.0002, n=3 AML, n=2 NK donors) (Fig 1E). Preliminary data shows simultaneous KO of the NK cell checkpoint cytokine-inducible SH2 containing protein (CISH) using CRISPR/Cas9 while maintaining efficient transposition (Fig 1F).Conclusions:We describe a novel, non-viral approach to CAR-NK cell production using the TcB DNA transposon system, supported by a clinically validated, GMP grade, irradiated, EBV-LCL with clinical scale expansion capability. Purified CAR-NK cell populations were achieved by immunomagnetic sorting without requiring a selection marker or cytotoxic exposure. Preliminary data supports the ability to simultaneously perform CRISPR/Cas9 gene editing - in this case applied to KO of the CISH gene, an NK cell checkpoint with multiple established benefits, including enhanced in vivo NK cell persistence and improved metabolic health. Manufactured using this process, we also present the first reported pre-clinical activity of a CLL-1 CAR-NK cell therapy in AML, demonstrating enhanced targeting of populations enriched for LSC. [Display omitted] DisclosuresGurney: ONK Therapeutics: Research Funding. O'Reilly: ONK Therapeutics: Research Funding. Corcoran: ONK Therapeutics: Current Employment. Brophy: ONK Therapeutics: Current Employment; Autolus: Ended employment in the past 24 months. Hardwicke: ONK Therapeutics: Current Employment; Novartis: Ended employment in the past 24 months. Hermanson: Bio-Techne: Current Employment. Szegezdi: ONK Therapeutics: Research Funding. O'Dwyer: Janssen: Consultancy; ONK Therapeutics: Current Employment, Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Research Funding.

Promising Preliminary Activity of Optimized Affinity, CD38 CAR NK Cells Generated Using a Non-Viral Engineering Approach in Gene Edited Cord Blood Derived NK Cells for the Treatment of Multiple Myeloma

CD38 is a multifunctional cell surface protein that is frequently overexpressed on malignant plasma cells as well as on immune suppressive cells within the tumor microenvironment and constitutes a validated immunotherapeutic target in the treatment of multiple myeloma (MM). At ONK Therapeutics we are developing a gene edited, cord blood-derived NK (CBNK) cell product targeting CD38 for treatment of patients with relapsed and/or refractory MM.The product will be generated using a workflow shown in Figure 1A. This involves starting with cord blood that is processed for NK expansion using a clinically validated, Epstein Barr Virus-transformed lymphoblastoid cell line (EBV-LCL) feeder layer. The NK cells would undergo genetic engineering that involves gene editing followed by a non-viral chimeric antigen receptor (CAR) introduction process mediated by the TcBuster (TcB) DNA transposon system (Biotechne). This is followed by a second round of expansion on the EBV-LCL feeder layer resulting in a characterized NK cell product that can then be cryopreserved.In order to develop protocols for optimizing the best transfection efficiencies using the Maxcyte ATx instrument, GFP mRNA (TriLink) was used for transfecting CBNK cells using different electroporation programs. High transfection efficiency was obtained using all programs (Figure 1B.), with the best from program NK4.Since the product employs an optimized affinity second generation anti CD38 CAR (Stikvoort et al., Hemasphere 2021) which could also target CD38 expressed on neighbouring activated NK cells, it is imperative to knock out (KO) the cell surface expression of CD38 on the CAR-NK cells. To achieve this we carried out CRISPR Cas9 based KO studies of CD38 (Figure 1C. left top), using guide RNAs targeting CD38 (Synthego) in the form of a ribonucleoprotein (RNP) complex with Cas9. CBNK cells were transfected using the Maxcyte ATx instrument and CD38 cell surface expression monitored. As shown in Figure 1C. (left top), complete CD38 KO was achieved 11 days post transfection.ONK Therapeutics is actively involved in targeting and downregulating the negative regulator of cytokine signalling, cytokine inducible SH2-containing protein (CIS), which is encoded by the CISH gene, as part of their CBNK products. It has been demonstrated that in addition to facilitating greater cytokine signalling, CISH KO also confers greater metabolic capacity to NK cells resulting in their increased persistence (Daher et al., Blood 2021). Therefore, ONK Therapeutics have evaluated CISH KO in CBNK cells (Figure 1C, top right) using the same scheme that was used for the CD38 KO. Guide RNAs in the form of a RNP complex with Cas9 (Synthego) were transfected into CBNK cells and intracellular CIS protein levels monitored over time. Almost complete KO was attained by 9 days post transfection. In order to dial in CISH KO as part of the product, we further carried out a simultaneous KO of CD38 and CISH, in addition to individual KO of CD38 or CISH (Fig 1C, bottom). Simultaneous multiplexing of the CD38 and CISH KOs resulted in efficient double KO (DKO) . The extent of knock down leading to KO in the DKO setting was very similar to that of individual gene KOs.We then introduced the anti CD38 CAR as part of a transposon that could be transposed by TcB transposase in CBNK cells. After DKO of CD38 and CISH in CBNK cells, the transposon DNA and mRNA for transposase were electroporated. CAR expression was detected 4-5 days post transposition (Figure 1D) with more than 50% of cells expressing the anti CD38 CAR. These CAR expressing CBNK cells were then tested for functionality in a co-culture kill assay against the CD38 positive MM cell line, RPMI8226, which was engineered to express firefly luciferase. In a 4 hour killing assay, robust killing of the RPMI8226 cells was achieved by the CAR-CBNK cells with an EC 50 ten times lower (more potent) than that of mock electroporation control CBNK cells.To our knowledge this is the first successful expression of an anti CD38 CAR in cord-derived NK cells, and with a double CD38/CISH KO, using non-viral CAR insertion approaches. Current work is focusing on designing and developing a manufacturing-ready workflow for this potential product and further examining the effects of CAR NK cell activity in a DKO setting where both KOs contribute to improved metabolism and potentially NK cell persistence, as well as exploring the added benefit of a DR5 TRAIL variant to enhance cytotoxicity. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Identification and characterization of a novel Epstein-Barr Virus-encoded circular RNA from LMP-2 Gene

Epstein-Barr virus (EBV) has been recently found to generate novel circular RNAs (circRNAs) through backsplicing. However, comprehensive catalogs of EBV circRNAs in other cell lines and their functional characterization are still lacking. In this study, we have identified a list of putative EBV circRNAs in GM12878, an EBV-transformed lymphoblastoid cell line, with a significant majority encoded from the EBV latent genes. A novel EBV circRNA derived from the exon 5 of LMP-2 gene which exhibited highest prevalence, was further validated using RNase R assay and Sanger sequencing. This circRNA, which we term circLMP-2_e5, can be universally detected in a panel of EBV-positive cell lines modelling different latency programs. It ranges from lower expression in nasopharyngeal carcinoma (NPC) cells to higher expression in B cells, and is localized to both the cytoplasm and the nucleus. We provide evidence that circLMP-2_e5 is expressed concomitantly with its cognate linear LMP-2 RNA upon EBV lytic reactivation, and may be produced as a result of exon skipping, with its circularization possibly occurring without the involvement of cis elements in the short flanking introns. Furthermore, we show that circLMP-2_e5 is not involved in regulating cell proliferation, host innate immune response, its linear parental transcripts, or EBV lytic reactivation. Taken together, our study expands the current repertoire of putative EBV circRNAs, broadens our understanding of the biology of EBV circRNAs, and lays the foundation for further investigation of their function in the EBV life cycle and disease development.

Cell-cycle-dependent EBNA1-DNA crosslinking promotes replication termination at oriP and viral episome maintenance

Epstein-Barr virus (EBV) is an oncogenic human herpesvirus that persists as a multicopy episome in proliferating host cells. Episome maintenance is strictly dependent on EBNA1, a sequence-specific DNA-binding protein with no known enzymatic activities. Here, we show that EBNA1 forms a cell cycle-dependent DNA crosslink with the EBV origin of plasmid replication oriP. EBNA1 tyrosine 518 (Y518) is essential for crosslinking to oriP and functionally required for episome maintenance and generation of EBV-transformed lymphoblastoid cell lines (LCLs). Mechanistically, Y518 is required for replication fork termination at oriP invivo and for formation of SDS-resistant complexes invitro. EBNA1-DNA crosslinking corresponds to single-strand endonuclease activity specific to DNA structures enriched at replication-termination sites, such as 4-way junctions. These findings reveal that EBNA1 forms tyrosine-dependent DNA-protein crosslinks and single-strand cleavage at oriP required for replication termination and viral episome maintenance.

Generation and reactivity analysis of human recombinant monoclonal antibodies directed against epitopes on HLA-DR.

In kidney transplantation, eplet mismatches between donor and recipient have been associated with de novo donor‐specific antibody development. Eplets are theoretically defined configurations of polymorphic amino acids and require experimental verification to establish whether they can be bound by alloantibodies. Human HLA‐specific monoclonal antibodies (mAbs) have been instrumental for this purpose but are largely lacking for HLA class II. In this study, we isolated single HLA‐DR‐specific memory B cells from peripheral blood of immunized individuals (n = 3) using HLA class II tetramers to generate recombinant human HLA‐DR antigen‐reactive mAbs (n = 5). Comparison of the amino acid composition of the reactive HLA alleles in relation to the antibody reactivity patterns led to identification of 3 configurations, 70Q 73A, 31F 32Y 37Y, and 14K 25Q recognized, respectively, by HLA‐DRB1*01:01, HLA‐DRB1*04:01, and HLA‐DRB1*07:01 antigen‐reactive mAbs. The first 2 correspond to eplets 70QA and 31FYY and can now be considered antibody verified. The latter indicates that eplet 25Q needs to be redefined before being considered as antibody verified. Generation and reactivity analysis of human HLA‐DR mAbs allowed for identification of amino acid configurations corresponding to known eplets, whereas the other patterns may be used to redefine eplets with similar, but not identical predicted amino acid composition.

Transgenic lymphocytes targeting minor histocompatibility antigens for post-transplant immunotherapy

Background & Aim The relapse of acute leukemia occurs in one third of the patients after HLA-matched allogeneic stem cell transplantation (HSCT), resulting in high mortality. Disparities in minor histocompatibility antigens (MiHA, MHC-presented peptides derived from polymorphic self-proteins) can result in the development of allogeneic immune response. Several MiHA (HA-1, HA-2, and ACC-1Y) are encoded by the genes predominantly expressed in hematopoietic tissues. They represent promising targets for post-HSCT relapse therapy. The immune response towards them selectively targets patient hematopoietic cells sparing hematopoietic cells of donor origin and patients healthy tissues. Methods, Results & Conclusion Naïve CD8+ T cells from MiHA-negative donors were expanded in vitro using autologous dendritic cells, pulsed with MiHA peptides. Antigen-specific cells were sorted, and the sequences of T-cell receptor (TCR) alpha and beta chains were determined by NGS. 8, 10, and 1 TCRs specific for HA-1, HA-2, and ACC-1Y antigens, respectively, were cloned in the lentiviral vectors containing coreceptor CD8. T cell lines were generated for further analysis. To prevent off-target effects caused by the mispairing of endogenous and recombinant TCR chains, the endogenous TCR knockout was made using gRNA/Cas9 ribonucleoprotein complexes. Transgenic alpha and beta-chain constant domains were modified to be resistant to Cas9 cleavage and to form more stable TCR. Antigen-specificity was measured by binding to MHC-tetramers loaded with the MiHA peptide and by TCR-mediated activation of the Jurkat 76 triple parameter reporter cell line upon antigen stimulation. Most of the generated cell lines were specific to the desired antigen, and none were activated in response to irrelevant peptides presented in the same HLA. After analysis of cross-reactivity to allogeneic HLA on the panel of Epstein-Barr virus-transformed lymphoblastoid cell lines, the best receptors would be selected for further therapeutic application. Adoptive transfer of MiHA-specific genetically modified T cells holds great promise for the treatment of leukemia relapse after allo-HSCT. The work was supported by the Russian Foundation for Basic Research grant 19-29-04156. The relapse of acute leukemia occurs in one third of the patients after HLA-matched allogeneic stem cell transplantation (HSCT), resulting in high mortality. Disparities in minor histocompatibility antigens (MiHA, MHC-presented peptides derived from polymorphic self-proteins) can result in the development of allogeneic immune response. Several MiHA (HA-1, HA-2, and ACC-1Y) are encoded by the genes predominantly expressed in hematopoietic tissues. They represent promising targets for post-HSCT relapse therapy. The immune response towards them selectively targets patient hematopoietic cells sparing hematopoietic cells of donor origin and patients healthy tissues. Naïve CD8+ T cells from MiHA-negative donors were expanded in vitro using autologous dendritic cells, pulsed with MiHA peptides. Antigen-specific cells were sorted, and the sequences of T-cell receptor (TCR) alpha and beta chains were determined by NGS. 8, 10, and 1 TCRs specific for HA-1, HA-2, and ACC-1Y antigens, respectively, were cloned in the lentiviral vectors containing coreceptor CD8. T cell lines were generated for further analysis.

Interindividual Differences in DNA Adduct Formation and Detoxification of 1,3-Butadiene-Derived Epoxide in Human HapMap Cell Lines.

Smoking-induced lung cancer is a major cause of cancer mortality in the US and worldwide. While 11-24% of smokers will develop lung cancer, risk varies among individuals and ethnic/racial groups. Specifically, African American and Native Hawaiian cigarette smokers are more likely to get lung cancer as compared to Caucasians, Japanese Americans, and Latinos. It is important to identify smokers who are at the greatest risk of developing lung cancer as they should be candidates for smoking cessation and chemopreventive intervention programs. Among 60+ tobacco smoke carcinogens, 1,3-butadiene (BD) is one of the most potent and abundant (20-75 μg per cigarette in mainstream smoke and 205-361 μg per cigarette in side stream smoke). BD is metabolically activated to 3,4-epoxy-1-butene (EB), which can be detoxified by glutathione S-transferase theta 1 (GSTT1)-mediated conjugation with glutathione, or can react with DNA to form 7-(1-hydroxy-3-buten-2-yl)guanine (EB-GII) adducts. In the present study, we employed EBV-transformed human lymphoblastoid cell lines (HapMap cells) with known GSTT1 genotypes to examine the influence of the GSTT1 gene on interindividual variability in butadiene metabolism, DNA adduct formation/repair, and biological outcomes (apoptosis). We found that GSTT1- HapMap cells treated with EB in culture produced lower levels of glutathione conjugates and were more susceptible to apoptosis but had similar numbers of EB-GII adducts as GSTT1+ cells. Our results suggest that GSTT1 can influence an individual's susceptibility to butadiene-derived epoxides.

B cells infected with Type 2 Epstein-Barr virus (EBV) have increased NFATc1/NFATc2 activity and enhanced lytic gene expression in comparison to Type 1 EBV infection.

Humans are infected with two distinct strains (Type 1 (T1) and Type 2 (T2)) of Epstein-Barr virus (EBV) that differ substantially in their EBNA2 and EBNA 3A/B/C latency genes and the ability to transform B cells in vitro. While most T1 EBV strains contain the “prototype” form of the BZLF1 immediate-early promoter (“Zp-P”), all T2 strains contain the “Zp-V3” variant, which contains an NFAT binding motif and is activated much more strongly by B-cell receptor signalling. Whether B cells infected with T2 EBV are more lytic than cells infected with T1 EBV is unknown. Here we show that B cells infected with T2 EBV strains (AG876 and BL5) have much more lytic protein expression compared to B cells infected with T1 EBV strains (M81, Akata, and Mutu) in both a cord blood-humanized (CBH) mouse model and EBV-transformed lymphoblastoid cell lines (LCLs). Although T2 LCLs grow more slowly than T1 LCLs, both EBV types induce B-cell lymphomas in CBH mice. T1 EBV strains (M81 and Akata) containing Zp-V3 are less lytic than T2 EBV strains, suggesting that Zp-V3 is not sufficient to confer a lytic phenotype. Instead, we find that T2 LCLs express much higher levels of activated NFATc1 and NFATc2, and that cyclosporine (an NFAT inhibitor) and knockdown of NFATc2 attenuate constitutive lytic infection in T2 LCLs. Both NFATc1 and NFATc2 induce lytic EBV gene expression when combined with activated CAMKIV (which is activated by calcium signaling and activates MEF2D) in Burkitt Akata cells. Together, these results suggest that B cells infected with T2 EBV are more lytic due to increased activity of the cellular NFATc1/c2 transcription factors in addition to the universal presence of the Zp-V3 form of BZLF1 promoter.

PS1314 ISOLATION AND VALIDATION OF THE FIRST FUNCTIONAL HLA‐A∗01:01‐RESTRICTED EBV‐LMP2‐SPECIFIC T‐CELLS FOR TREATMENT OF EBV‐ASSOCIATED TYPE II/III LYMPHOMAS

Background:Epstein Barr virus (EBV) is associated with the development of a broad range of malignancies, including Burkitt's lymphoma, Hodgkin and non‐Hodgkin lymphomas, post‐transplant lymphoproliferative disorder (PTLD), nasopharyngeal carcinoma and gastric carcinoma. Although many EBV‐associated lymphomas only express weak immunogenic EBV antigens (e.g. EBNA1 and BARF1), lymphomas with type II or III latency often express immunogenic LMP1 and LMP2. Different strategies have been developed to manufacture EBV‐LMP1/2‐specific T‐cell products for clinical application and potential effectivity of autologous and donor‐derived EBV‐LMP1/2‐specific T‐cells has been demonstrated in clinical trials. Surprisingly, to date, no EBV‐specific T‐cells recognizing a peptide in the common HLA‐A∗01:01 allele have been found, while an HLA‐A∗01:01‐associated risk for EBV+ Hodgkin lymphomas has been reported.Aims:A need thus exists for HLA‐A∗01:01‐restricted EBV‐specific T‐cell products, especially EBV‐LMP1/2 specific T‐cells.Methods:Based on MHC‐class‐I peptide predictions, HLA‐A∗01:01‐binding peptides derived from different immunogenic EBV antigens were identified, and HLA‐A∗01:01/peptide tetramer complexes were synthesized (EBNA3A‐YTD, EBNA3A‐FLQ, BZLF1‐FTP, LMP2‐ESE, LMP2‐LTE). For these 5 specificities, tetramer positive CD8 T‐cells were sorted by flow cytometry from peripheral blood mononuclear cells (PBMC) of 6 HLA‐A∗01:01 positive healthy donors. Specificity of expanded T‐cells was confirmed by tetramer staining. Functional avidity was assessed using TAP2‐deficient T2 cells transduced with HLA‐A∗01:01 and exogenously loaded with specific peptide. The recognition of endogenously processed and presented antigen was analyzed using K562 cells transduced with HLA‐A∗01:01 and a retroviral vector encoding the full protein sequence of LMP2, HLA‐A∗01:01‐positive EBV transformed lymphoblastoid cell lines (EBV‐LCLs) and IFNy ELISA as read‐out.Results:HLA‐A∗01:01‐restricted EBV‐specific T‐cells were present at low frequencies in total PBMCs from all 6 donors. After flow cytometric cell sorting, only EBV‐LMP2‐ESE‐specific T‐cells of 5/6 donors expanded, resulting in pure tetramer+ CD8 T‐cell lines. Analysis of the T‐cell receptor beta chain (TCR‐Vβ) usage showed that these EBV‐LMP2‐ESE specific T‐cell populations were oligoclonal. To further analyze their functional avidity, EBV‐LMP2‐ESE‐specific T‐cells were tested against HLA‐A∗01:01‐transduced TAP2‐deficient T2 cells exogenously loaded with peptide. Four out of 5 isolated T‐cell populations recognized T2 cells loaded with 1–103nM peptide, indicating intermediate to high avidity. EBV‐LMP2‐ESE‐specific T‐cells were subsequently tested against HLA‐A∗01:01/LMP2‐transduced K562 cells. All functional EBV‐LMP2‐ESE‐specific T‐cell populations could recognize these transduced K562 cell lines, indicating that the EBV‐LMP2‐ESE peptide is successfully processed, presented and recognized by EBV‐LMP2‐ESE‐specific T‐cells. Recognition of HLA‐A∗01‐positive EBV‐LCLs further illustrated the potential of the EBV‐LMP2‐ESE‐specific T‐cells to recognize endogenous LMP2.Summary/Conclusion:We describe the isolation and validation of the first functional HLA‐A∗01:01‐restricted EBV‐LMP2‐specific T‐cell populations, which can be used for adoptive transfer to treat EBV‐associated type II/III lymphomas, malignancies of epithelial origin and PTLDs. Despite the very low frequency of these T‐cells, we were able to obtain these T‐cells from 5 out of 6 donors and showed their capacity to recognize both exogenously loaded as endogenously processed and presented EBV‐LMP2‐ESE peptide.

Human natural killer cell manufacturing using a closed system process for patients with metastatic solid tumors or hematologic malignancies

Background & Aim As potent effectors of the innate immune system natural killer (NK) cells are currently utilized in clinical trials world-wide to treat a variety of hematologic and solid organ malignancies. The ongoing phase I/II study at the National Institutes of Health (08-H-0186) previously utilized open-system processing requiring 60-80 small GREX flasks and single donor AB plasma in the culture medium. We optimized the manufacturing process to generate large numbers of NK cells using closed-system processing with large 5 liter GREX containers and commercially available pooled AB serum. Methods, Results & Conclusion The manufacturing process entails two stages: Preparation of the feeder EBV-transformed lymphoblastoid cell line (LCL) and NK cell culture. LCL are cultured in closed GREX100-CS vessels with XVIVO15 culture media containing 15mM HEPES, 2mM glutamax and 10% of either commercially available pooled AB serum or single donor AB plasma. With one culture vessel capable of generating 8-10 × 108 LCL feeder cells using 600-800mL of culture medium, 3 vessels sufficiently support a full scale culture. LCL number and viability are similar between groups containing serum vs. plasma. In the second stage, NK cell culture is initiated using 3 GREX500-CS with 40 × 106 NK cells and 400 × 106 irradiated LCL feeder cells in 500mL/vessel. Fresh XVIVO20 containing 15mM HEPES, 2mM glutamax, 500IU/mL IL2 and either 10% AB plasma or pooled AB serum is added on days 4, 8, 12 and 17. On day 14, a portion of the cells are harvested and washed for fresh infusion, and the remaining cells are re-seeded into a new vessel for harvest for second infusion on day 19. Day 14 and day 19 harvest generated 10-14 × 109 (1 vessel) and 50-70 × 109 NK cells (4-5 vessels) for fresh infusion, enough to support the target dose level of 1 × 108 (1st harvest) and 5 × 108 (2nd harvest) NK cells/kg. We found no significant differences in cell growth, viability or TRAIL expression when using AB plasma vs. AB serum. However, TRAIL expression was significantly increased when the concentration of the NK cells was kept below 4 × 106 cells/mL during culture. NK cells also demonstrated tumor cell line killing ability in vitro and had increased CD107. In summary, we have developed a new process that improves safety and consistency for culturing NK cells in large numbers for an ongoing clinical trial at the NIH. This process can be easily adapted by cell therapy manufacturing facilities at most academic institutions.

Gene expression changes in lymphoblastoid cell lines and primary B cells by dexamethasone.

Human Epstein-Barr virus-transformed lymphoblastoid cell lines (LCLs) have been thought to be a useful model system for pharmacogenomics studies. The purpose of this study was to determine the effect of Epstein-Barr virus transformation on gene expression changes by dexamethasone (Dex) in LCLs and primary B cells (PBCs) derived from the same individuals. We prepared LCLs and purified PBCs from the same six male donors participating in the Childhood Asthma Management Program clinical trial, and compared mRNA profiles after 6 h incubation with Dex (10 mol/l) or sham buffer. We assessed differential expression and put the list of differentially expressed genes into the web interface of ConsensusPathDB to find the pathway-level interpretation of our genes specified. As a supplementary analysis, we looked at the expression of the Dex-regulated (inducing or repressing) genes in treatment-naive PBCs and LCLs (pre-Dex treatment) from the GSE30916 dataset. By hierarchical clustering, we found clustering of probes by cell types but not by individuals irrespective of Dex treatment. We observed that the Dex-regulated genes significantly overlapped in PBCs and LCLs. In addition, the expression of these genes showed significant correlations between treatment-naive PBCs and LCLs. Common genes showing significantly decreased expressions by the Dex treatment in both cells were enriched in immune responses and proinflammatory signaling pathways. Taken together, these results suggest the uses of LCLs are representative of the primary biologic effects of corticosteroids treatment.