EBV-transformed B-cell Lines Research Articles

T-cell mitochondrial dysfunction and lymphopenia in DOCK2-deficient patients

This study delineates the mitochondrial defects in DOCK2-deficient T cells contributing to the T cell lymphopenia characteristic of this primary immunodeficiency.

A cancer-associated Epstein-Barr virus BZLF1 promoter variant enhances lytic infection.

Latent Epstein-Barr virus (EBV) infection contributes to both B-cell and epithelial-cell malignancies. However, whether lytic EBV infection also contributes to tumors is unclear, although the association between malaria infection and Burkitt lymphomas (BLs) may involve excessive lytic EBV replication. A particular variant of the viral promoter (Zp) that controls lytic EBV reactivation is over-represented, relative to its frequency in non-malignant tissue, in EBV-positive nasopharyngeal carcinomas and AIDS-related lymphomas. To date, no functional differences between the prototype Zp (Zp-P) and the cancer-associated variant (Zp-V3) have been identified. Here we show that a single nucleotide difference between the Zp-V3 and Zp-P promoters creates a binding site for the cellular transcription factor, NFATc1, in the Zp-V3 (but not Zp-P) variant, and greatly enhances Zp activity and lytic viral reactivation in response to NFATc1-inducing stimuli such as B-cell receptor activation and ionomycin. Furthermore, we demonstrate that restoring this NFATc1-motif to the Zp-P variant in the context of the intact EBV B95.8 strain genome greatly enhances lytic viral reactivation in response to the NFATc1-activating agent, ionomycin, and this effect is blocked by the NFAT inhibitory agent, cyclosporine, as well as NFATc1 siRNA. We also show that the Zp-V3 variant is over-represented in EBV-positive BLs and gastric cancers, and in EBV-transformed B-cell lines derived from EBV-infected breast milk of Kenyan mothers that had malaria during pregnancy. These results demonstrate that the Zp-V3 enhances EBV lytic reactivation to physiologically-relevant stimuli, and suggest that increased lytic infection may contribute to the increased prevalence of this variant in EBV-associated malignancies.

Abstract 649: Development of a novel RNA sequencing approach that identifies aberrant splicing in cancer predisposing genes

Abstract Background: High-throughput sequencing efforts in molecular tumor diagnostics detect increased numbers of novel genetic variants, including the ones that are predicted to affect splicing. Current approach for predicting whether a specific variant may result in aberrant RNA transcripts often consists of in-silico prediction using bioinformatics prediction tools, which is sometimes followed by RT-PCR analysis of the RNA extracted from blood, or functional splicing reporter assays. Although high-quality patient RNA analysis is usually preferred, such RNA is not always available, or the analysis is hampered due to degradation of aberrant transcripts through nonsense-mediated decay. To overcome this issue, we investigated the possibility of analyzing RNA isolated from formalin-fixed paraffin-embedded (FFPE) tissues, with the underlying hypothesis that formalin fixation may inhibit RNA degradation, enabling the detection of aberrant RNA products. Methods: RNA analysis was performed for 13 canonical splice site variants predicted or known to affect splicing in the cancer predisposition genes MLH1, MSH2, MSH6, APC and BRCA1. Of 11 variants, total nucleic acid was obtained from FFPE tumor tissues. For two variants, no FFPE material was available, but EBV-transformed B-cells carrying a splice variant were cultured. Primers were designed to amplify the exon-exon boundary predicted to be affected. Housekeeping gene expression was assessed to determine presence of RNA. Results: Total nucleic acid was successfully isolated for 10 variants from eight FFPE tumor tissues and two EBV-transformed B-cell lines. Aberrant splicing was confirmed in all six variants known to result in splicing. Of one known splice variant, present in the B-cell line, the known aberrant splice product could only be detected after formalin fixation of the cells, suggesting that formalin fixation possibly inhibited RNA degradation. Conclusion: Aberrant splicing was successfully identified in all known splice variants and three of four variants predicted to affect splicing, of which the splice effect was previously unknown. Using a RNA sequencing approach, somatic splice variants could be easily and rapidly analyzed, enabling retrospective analysis of variants predicted to result in splicing when only FFPE material is available in clinical settings. Citation Format: Anne M. Jansen, Heleen M. van der Klift, Jaap D. van Eendenburg, Carli M. Tops, Juul T. Wijnen, Frederik J. Hes, Ajay Goel, Hans Morreau, Tom van Wezel. Development of a novel RNA sequencing approach that identifies aberrant splicing in cancer predisposing genes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 649.

Lentiviral Vectors Incorporating Ubiquitous Chromatin Opening Element Driving Canine CD18 Expression

Leukocyte adhesion deficiency type 1 (LAD1) in humans is caused due to mutations in the ITGB2 gene encoding the leukocyte CD18 subunit (b2 integrin). This results in defective leukocyte adhesion and migration leading to recurrent episodes of life-threatening bacterial infection. Canine leukocyte adhesion deficiency (CLAD) represents a disease-specific large animal model of LAD1 in which new therapeutic approaches could be tested. Our previous studies have demonstrated variable efficiency of CD18 expression under the control of several promoters. These include cellular promoters such as those of human elongation factor 1a (hEF1a): long (1169bp) and short (248bp) fragments, human phosphoglycerate kinase (hPGK), human CD11b and human CD18 genes. In addition, murine stem cell virus (MSCV) promoter has also been demonstrated to lead to very high levels of CD18 expression in CLAD CD34+ cells thereby reversing the CLAD phenotype in dogs previously treated with both foamy and lentiviral vectors. But, due to potential genotoxicity associated with the use of viral promoters, we continued our efforts in search of novel cellular promoters. One such promoter is the ubiquitous chromatin opening element (UCOE) from the human heterogeneous ribonucleoprotein A2/B1 and chromobox homolog 3 (HNRPA2B1-CBX3) loci. UCOE has been previously shown to display reproducible and stable transgene expression within the context of a self-inactivating (SIN) lentiviral vector in the absence of classical enhancer activity (Zhang et al., Blood 2007).It has also been shown to confer resistance to DNA methylation-mediated transgene silencing even upon integration into the heterochromatin regions of the host chromosome (Zhang et al., Mol Ther. 2010). Since the full-length element is about 2.6 kb, we cloned and tested different fragment lengths of the UCOE promoter in a SIN lentiviral vector (pCL20) in CLAD CD34+ cells in vitro. Efficiency of expression of CD18 obtained with the six promoter fragments of UCOE (in bp), namely U3'631, U3'1262, U3'652, U5'1357, U5'723 and U5'655 were compared to those obtained with an MSCV promoter. Functional viral titers were first determined using a human LAD EBV-transformed B-cell line that lacks endogenous human CD18. When comparable titers of each vector were used in an overnight transduction of CLAD CD34+ cells after a 24h cytokine prestimulation in vitro, the percentage of CD18+ cells 5 days after transduction were as follows: U3'631 - 8.49%, U3'1262 - 15.9%, U3'652 - 21.3% (tested at MOI 100), U5'1357 - 2.05% (tested at MOI 30), U5'723 - 2.44% (tested at MOI 20), U5'655 - 3.01% (tested at MOI 50) and MSCV - 35.3% (tested at MOI 100). The CD18 expression levels driven by some of these promoter fragments were comparable to those driven by cellular promoters mentioned previously. The UCOE is promising in that it could overcome possible gene silencing effects when used in vivo, unlike promoters such as EF1a and PGK which were largely subjected to post-transcriptional gene silencing with sub-therapeutic levels of CD18 as previously tested in the dog model. Hence, functional correction of the CD18 defect could be achieved with candidate UCOE-incorporating SIN lentiviral vector(s) when used in the treatment of CLAD dogs. DisclosuresNo relevant conflicts of interest to declare.

Abstract O.21: Inositol 1,4,5 triphosphate 3- kinase C regulates NLRP3 Inflammasome activation in Kawasaki disease

Background: ITPKC controls calcium homeostasis and was identified in genome-wide studies to be associated with susceptibility and severity of KD. NLRP3 is activated by danger signals, leading to inflammasome activation and release of IL-1beta and IL-18. Ca 2+ mobilization mediates NLRP3 activation. We studied the contribution of ITPKC and NLRP3 inflammasome activation in KD. Methods: Cytokine levels were measured using ELISA. Gene expression was assayed using Illumina HumanHT-12v4. EBV-transformed B-cell lines from KD patients with different ITPKC genotypes were used to study the gene and protein expression and Ca 2+ flux using qRT-PCR, Western blot analysis, and FACS respectively. In vivo and in vitro analyses from ITPKC-deficient and wildtype mice were performed using ELISA, spinning disc confocal and Westerns, to determine cytokine release, Ca 2+ flux and protein expression responses respectively. Results: Children with KD show increased circulating IL-1beta, IL-18, IL-1RA and IL18BP protein during acute phase of KD (KD n=48, febrile controls n=41 p<0.001). NLRP3 and its associated inflammasome complex, and the IL-1[[Unsupported Character - Symbol Font ]] and IL18 receptor genes were up-regulated nearly two fold during the acute KD compared to matched convalescent controls (n=171, p<0.001). Pathway analysis showed specific up-regulation of NLRP3 related genes. The KD-associated genetic polymorphism in ITPKC (rs28493229) is functional, directly modulating [Ca 2+ ] i mobilization resulting in NLRP3 activation and increased production of IL-1beta and IL-18, as demonstrated by gene expression, circulating protein levels and functional assays of PBMCs and immortalized cell lines from affected children. These biologic data are partnered with evidence showing resistance to IVIG therapy in those with the ITPKC polymorphism associated with the highest basal and stimulated [Ca 2+ ] i levels. Studies using ITPKC-deficient mice in an animal model of KD support the molecular, cellular and clinical findings in affected children. Significance: ITPKC regulates NLRP3 activation via control of [Ca 2+ ] i mobilization, directing production of IL-1beta and IL-18, pointing to the key role of calcium mobilization in the immunopathogenesis of KD.

Promiscuous T-cell responses to drugs and drug-haptens

Promiscuous T-cell responses to drugs and drug-haptens

Identification of susceptible HLA class II co‐amoxiclav genotypes based on the analysis of drug‐specific T‐cells from patients with liver injury

Drug-induced liver injury (DILI) is a major concern for public health. Several forms of DILI are associated with the expression of specific MHC genes suggesting that the adaptive immune system is involved in the disease pathogenesis. We have identified drug-responsive T-cells in patients with flucloxacillin induced DILI and shown that the drug antigen is presented to T-cells in the context of the HLA risk allele (HLA-B*5701). The combination of amoxicillin and clavulanic acid (coamoxiclav) is one of the most frequently prescribed drugs. Occasionally, human exposure is associated with DILI. Individual susceptibility can be influenced by HLA genotypes (e.g. DRB1*1501 risk; DRB1*07:01 risk) and we have recently characterized drug-specific T-cells in patients with co-amoxiclav-induced DILI. The objective of this study was to use amoxicillin-specific CD4+ T-cell clones from 2 DILI patients to investigate the mechanisms of drug antigen presentation and to identify susceptible HLA class II genotypes based on analysis of drug specific T-cell responses. Method Antigen presenting cells (APC) expressing different HLA alleles generated from our HLA-typed PBMC cell bank were used to investigate the latter. EBV-transformed B-cell lines from 15 donors expressing HLA alleles of interest were generated and used as APC. To characterize mechanism(s) of T-cell activation, APC were (1) omitted from the assay, (2) pulsed with amoxicillin for 1-16h and (3) treated with MHC blocking antibodies. Results

Loss-of-function of the protein kinase C δ (PKCδ) causes a B-cell lymphoproliferative syndrome in humans

AbstractDefective lymphocyte apoptosis results in chronic lymphadenopathy and/or splenomegaly associated with autoimmune phenomena. The prototype for human apoptosis disorders is the autoimmune lymphoproliferative syndrome (ALPS), which is caused by mutations in the FAS apoptotic pathway. Recently, patients with an ALPS-like disease called RAS-associated autoimmune leukoproliferative disorder, in which somatic mutations in NRAS or KRAS are found, also were described. Despite this progress, many patients with ALPS-like disease remain undefined genetically. We identified a homozygous, loss-of-function mutation in PRKCD (PKCδ) in a patient who presented with chronic lymphadenopathy, splenomegaly, autoantibodies, elevated immunoglobulins and natural killer dysfunction associated with chronic, low-grade Epstein-Barr virus infection. This mutation markedly decreased protein expression and resulted in ex vivo B-cell hyperproliferation, a phenotype similar to that of the PKCδ knockout mouse. Lymph nodes showed intense follicular hyperplasia, also mirroring the mouse model. Immunophenotyping of circulating lymphocytes demonstrated expansion of CD5+CD20+ B cells. Knockdown of PKCδ in normal mononuclear cells recapitulated the B-cell hyperproliferative phenotype in vitro. Reconstitution of PKCδ in patient-derived EBV-transformed B-cell lines partially restored phorbol-12-myristate-13-acetate–induced cell death. In summary, homozygous PRKCD mutation results in B-cell hyperproliferation and defective apoptosis with consequent lymphocyte accumulation and autoantibody production in humans, and disrupts natural killer cell function.

Advances in basic and clinical immunology in 2012

Basic and clinical immunology articles published in the Journal in 2012 were mostly related to the expanding area of primary immunodeficiencies (PIDs). Novel forms of PID were identified by using whole-exome sequencing or after careful examination of flow cytometric data, as in the reports of lymphocyte-specific protein tyrosine kinase, CD27, and CD21 deficiencies. Absent IgG and IgA memory B cells were described in patients with hyper-IgE syndrome, which is consistent with defective antibody response and suggests a potential benefit of immunoglobulin replacement. Impaired production of antibodies to polysaccharide antigens by the human B-cell subset analog to murine B-1 cells was reported in a child with selective polysaccharide antibody deficiency. Increased production of inflammatory cytokines by monocyte-derived cells on Toll-like receptor activation was reported in patients with X-linked agammaglobulinemia, underscoring the important role of Bruton tyrosine kinase in modulation of inflammation. The mechanisms explaining susceptibility to yeast infections and development of chronic mucocutaneous candidiasis were extensively studied. Universal newborn screening for T-cell deficiencies is being implemented in several states, resulting in the diagnosis of a higher number of immunodeficient newborns than previously estimated. The use of laboratory testing to distinguish PIDs from HIV infection was clarified. In the management of PIDs, refinement of indication and strategies to hematopoietic stem cell transplantation resulted in improved outcomes. The use of anti-IL-6 mAbs showed promise as an alternative treatment in patients with Schnitzler syndrome.

CD23 molecule acts as a galactose-binding lectin in the cell aggregation of EBV-transformed human B-cell lines

Epstein-Barr virus (EBV)-transformed human B-cell lines, L-KT9 and DH3 cells express CD23 antigen, and grow in a mixture of single and aggregated cells. The CD23 molecule has high amino acid sequence homology with C-type lectin and recently we have shown that the solubilized CD23 molecule can really interact with galactose residues on glycoproteins. In this study, therefore, we tested whether CD23 antigen on the cell surface really acts as a galactose-binding lectin in the aggregation of these cells. The EBV-transformed cells (L-KT9) were separated into an aggregated-cell-rich fraction and a single-cell-rich fraction. Aggregated cells disaggregated after removal of galactose by beta-galactosidase treatment, whereas single cells made large aggregation on sialidase treatment, and this aggregation was inhibited in the presence of asialo-fetuin. On the other hand, naturally aggregated cells become single cells with anti-CD23 monoclonal antibody (mAB) as well as the soluble form of CD23, but not with anti-CD21 mAB. In addition, L-KT9 and DH3 cells bound to asialo-fetuin-coupled Sepharose (ASF-Sepharose) and this binding was significantly inhibited by pre-treatment of cells with anti-CD23, but not with anti-CD21 or other anti-adhesion molecules. From these results, we conclude that the naturally aggregated state of EBV-transformed cells occurs mainly through the interaction of CD23 as a lectin molecule and galactose residues as its ligand.

Moving Successful Virus-specific T-cell Therapy for Hematopoietic Stem Cell Recipients to Late Phase Clinical Trials

There are now multiple effective strategies for the activation and expansion, or direct selection of virus-specific T cells that may be able to eliminate a range of virus infections in the immunocompromised host. In this mini-review, we explain how the stage is set for their rigorous evaluation in large-scale clinical trials. Virus infections are the cause of 30% or more of transplant-related deaths in recipients of T-cells depleted, allogeneic hematopoietic stem cell transplants (HSCT).1 Epstein-Barr virus (EBV), cytomegalovirus (CMV), and adenoviruses (AdV) are the most common culprits, but other common viruses like parainfluenza virus, respiratory syncytial virus, influenza viruses, polyomaviruses, and human herpesvirus 6 together contribute significant morbidity and mortality (Figure 1).2,3,4 Small molecule therapies are often ineffectual, always costly and frequently produce significant adverse effects. Figure 1 Relative frequency of viral infections after HSCT. Adv, adenovirus; BK, JC, KI, and WU (polyomaviruses); CMV, cytomegalovirus; H1N1, influenza strain Hemagglutinnin 1 Neuraminidase 1); HHV, human herpes virus; HSCT, hematopoietic stem cell transplant; ... Virus-specific T-cells derived from stem cell donors can prevent and treat post-transplant viral infections, in the recipients for whom they were intended, and also in partially human leukocyte antigen (HLA)-matched, third party recipients.5,6,7 The low toxicity and long-term protection provided by virus-specific T-cells compares favorably with the significant toxicities and short-term effects of most antivirals.5,6,8,9,10 Is it time, therefore, to begin the transfer of T-cell manufacturing from academic phase I/II clinical trials into hospital or industry-supported facilities so that virus-specific T-cells can be made available to all high-risk HSCT recipients? Several barriers prevent the broader use of virus-specific T-cell therapies after stem cell transplantation. While T-cell therapies for EBV, CMV, and AdV have clearly demonstrated their safety and efficacy both as prophylaxis and as therapy, for many other viruses, the antigens that induce protective T-cells have yet to be identified. Moreover, these infections may occur in <5% of patients making it difficult to perform the rigorous comparative effectiveness studies that will be required to show lower overall cost, fewer adverse effects, and equivalent or superior efficacy. Before any of these barriers can be breached effectively by the academic institutions who are the major developers of these T-cells therapeutics, we must select and optimize manufacturing strategies that are robust and scalable and have the lowest possible cost. Finally, the leap to late phase trials cannot be accomplished by academic institutions alone, but requires partnership with industry. This article will largely deal with selection and optimization of virus-specific T-cell manufacturing strategies.

Detection and Characterization of Antigen-Driven CD8+ T Lymphocytes Specific for Wilms' Tumor Antigen in Patients with Non-Hodgkin Lymphoma

Abstract 953 Wilms' tumor antigen (WT1)is expressed in many solid tumors and hematologic malignancies and serves a physiological role in the maintenance of the tumorigenic phenotype. Immunologic recognition of WT1 has been observed in patients with myeloid and lymphoid leukemia, solid tumors, and even in healthy adults. However, little is known about WT1 expression or WT1-specific immune responses in patients with non-Hodgkin lymphoma (NHL). Current therapeutic modalities for NHL patients are predominantly based on the highly successful application of the anti-CD20 mAb (Rituximab™) or radioactive derivatives (Zevalin™ or Bexxar™) combined with standard chemotherapy. Recent approaches have focused on generating NHL-specific idiotypes as an example of personalized medicine. However, harnessing a T cell response to malignant B or T cells in NHL patients has not succeeded, despite its attractiveness as a universal therapeutic option. We developed a detection system for WT1-specific T cells that is HLA independent by constructing a peptide library of 15mers (11 residues overlapping) corresponding to full length WT1 antigen. We analyzed peripheral blood mononuclear cells (PBMC) after priming with the peptide library for immune responses to the WT1 antigen detectable by flow cytometry based on the CD137 activation marker. Sixty-three NHL patients and 30 healthy donors (HD) were recruited under IRB-approved protocols. The cohort included patients who received allogeneic stem cell transplantation (alloHCT: n=42) or chemotherapy/autologous HCT (n=21). We observed that WT1-specific ex vivo CD8+ T cell responses were significantly greater in our NHL patient cohort (median 0.035%) compared to our randomly chosen HD cohort (0%, p=0.0013)(Figure 1). These WT1-specific CD8+CD137+ T cells co-expressed CD45RA and CD57 surface markers, consistent with them being a TEMRA (Terminal Effector Memory, RA+) population of highly differentiated T cells. Twenty-four of 43 patients (55.8%) with high/intermediate-grade NHL had a positive CD8+ T cell response (CD8+CD137+ ≥0.05%) to the WT1 antigen compared to four of 20 patients (20%) with low-grade NHL (p=0.011). None of the other clinical variables such as patient age, treatment modality (alloHCT vs. autoHCT or chemotherapy), disease status (CR vs. non-CR), prior acute GVHD (only in alloHCT, grade 0-I vs. II–IV) were significantly associated (p≤0.05) with WT1-specific CD8+ T-cell response in NHL patients when we conducted a multivariate analysis. PBMC from one HD and 12 NHL patients were expanded using in vitro stimulation (IVS) with the WT1 peptide library. All PBMC samples expanded in culture after 10–14 days by single IVS (median 7-fold expansion). We observed significantly higher IFN-γ responses in CD8+ T cells when IVS cells were re-stimulated with the WT1 library (median=3.0%, range: 0.08–5.5%) compared with the medium control (median=0.39%, range: 0.05–1.7%, p=0.002). Remarkably, using standard in vitro techniques, the expanded PBMC were cytotoxic to WT1-peptide library-loaded T2 cells and autologous EBV-transformed B-cell lines derived from patients enrolled in the study. These results challenge the notion that central tolerance mechanisms eliminate high affinity WT1-specific T cells that kill tumor cells.In order to distinguish whether WT1-specific T cells are maintained in NHL patients because of an aberration of tolerance mechanisms or the presence of WT1 antigen as a stimulator, we used sensitive real time quantitative PCR (RT-qPCR) methods to detect transcripts in LN from NHL patients and HD. We succeeded in detecting WT1 mRNA transcripts in diseased lymph node (LN) tissues of NHL patients utilizing RT-qPCR technology. There was significantly greater WT1 mRNA in NHL LN (n=6) with an average copy number ratio of 39 (WT1/c-ABL × 104) compared with 0.66 in non-disease LN obtained from individuals without a cancer diagnosis (n=7, p<0.05). This exciting result strongly implicates WT1 antigen expression as a driving force of WT1-specific T cell expansion in NHL patients. In summary, ex-vivo WT1-specific CD8+ T cell responses can be detected in NHL patients, expand in vitro, produce IFN-γ, and are cytotoxic. These results are the first demonstration of significant T cell reactivity against the WT1 antigen in NHL patients. The potential for developing the WT1 antigen as a target for immunotherapy in NHL patients deserves further exploration. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

TCL1: A Shared Tumor-Associated Antigen for Immunotherapy Against B-Cell Lymphomas

Abstract 105Rituximab-based chemotherapy regimens have improved complete response rates, progression-free survival, and overall survival of B-cell non-Hodgkin lymphoma patients. However, most patients relapse and die of their lymphoma. To further improve clinical outcome, novel strategies that eradicate minimal residual disease (MRD) after induction therapy are needed. Therapeutic vaccines may induce antitumor antibody and T-cell responses and may eradicate MRD by complementary mechanisms. A recent randomized phase III clinical trial showed that patient-specific idiotype vaccination improves disease-free survival when administered in the setting of MRD in follicular lymphoma (FL), providing proof of principle that therapeutic vaccines can improve clinical outcome (Schuster et al, J Clin Oncol, 2011). However, generation of a custom-made vaccine formulation for each patient can be expensive and time consuming. To overcome these difficulties, identification of novel shared lymphoma-associated antigens is necessary.The T-cell leukemia/lymphoma 1 (TCL1) oncoprotein encoded by the TCL1 gene is a co-activator of Akt and promotes cell proliferation and survival. Ectopic expression of TCL1 in B cells in transgenic mice results in the development of B-cell malignancies and TCL1 was reported to be aberrantly expressed in multiple human B cell malignancies. Here, we determined whether TCL1 can serve as a novel shared tumor-associated antigen in B cell lymphomas. We analyzed the expression pattern of TCL1 in human B cell lymphomas and normal tissues by real-time PCR, flow cytometry, immunohistochemistry, and Western blotting. TCL1 mRNA transcripts were hyperexpressed in multiple types of primary B cell lymphomas [chronic lymphocytic leukemia (CLL), n=7; mantle cell lymphoma (MCL), n=6; FL, n=12, diffuse large B-cell lymphoma (DLBCL), n=5] as compared with B cells derived from normal donors. TCL1 mRNA was not detected in other normal tissues that included T cells, adipose tissue, esophagus, ovary, spleen, bladder, heart, placenta, brain, kidney, prostate, liver, muscle, thyroid, colon, lung, intestine, trachea, thymus, and cervix. Very low TCL1 mRNA was detected in testis. By flow cytometry, immunohistochemistry, and/or Western blotting we observed that the TCL1 protein is hyperexpressed in CLL, MCL, DLBCL, Burkitts lymphoma, and FL, but not splenic marginal zone lymphoma. To determine whether TCL1 is immunogenic, we synthesized overlapping 15-mer peptides spanning the entire length of the TCL1 protein and stimulated PBMC from HLA-A*0201+ (HLA-A2+) normal donors to generate peptide-specific T cells. We found that TCL165–79 peptide (TQIGPSLLPIMWQLY) consistently induced T cells that secreted significant amount of IFN-γ from 3 normal donors. By intracellular cytokine assay, we determined that the TCL165–79 peptide-specific IFN-g was produced by CD8+ T cells but not CD4+ T cells. Using a panel of peptide-pulsed EBV-transformed B-cell lines that were mismatched at the MHC Class I locus and MHC Class I blocking antibodies, we determined that HLA-A2 is the restriction element of the TCL165–79 peptide-specific T cells. Consistent with this, TCL165–79 peptide-specific CD8+ T cells lysed TCL1 expressing HLA-A2+ MCL cell lines but did not lyse, TCL1 expressing HLA-A2− myeloma cell line. Using a panel of peptides truncated progressively by one amino acid at the N- and C-termini of the TCL165–79peptide, we determined that TCL171–78(LLPIMWQL) is the minimal epitope that bound to HLA-A2. Furthermore, cytotoxic T lymphocytes (CTL) specific to this peptide efficiently killed lymphoma cell lines and primary lymphoma cells from CLL, MCL, DLBCL, and FL in an HLA-A2-restricted manner. Using TCL171–78 peptide-specific HLA-A2 tetramers, we found that TCL1 epitope-specific CTLs were present in the peripheral blood and/or tumor-infiltrating lymphocytes of B-cell lymphoma patients (n=8). More importantly, these TCL1 epitope-specific CTLs could be expanded in vitro and lysed autologous and allogeneic tumor cells in an HLA-A2-restricted manner. In conclusion, our results suggest that TCL1 is naturally processed and presented on the surface of primary lymphoma cells for recognition by CTL and can serve as a novel, shared tumor-associated antigen for therapeutic vaccine development against common B-cell lymphomas including CLL, MCL, DLBCL, and FL. Disclosures:Neelapu:Biovest International, Inc.: Research Funding.

Viral induction and targeted inhibition of galectin-1 in EBV+ posttransplant lymphoproliferative disorders

AbstractPosttransplant lymphoproliferative disorders (PTLDs) are potentially fatal, EBV-driven B-cell malignancies that develop in immunocompromised solid organ or hematopoietic stem cell recipients. In PTLD, the expression of EBV proteins, including latent membrane protein 1 (LMP1) and LMP2A, viral immune evasion strategies, and impaired host immune surveillance foster the proliferation of EBV-transformed B cells. Current PTLD treatment strategies include reduction of immunosuppression, which increases the risk of graft rejection, anti-CD20 treatment, combination chemotherapy, and administration of EBV-specific cytotoxic T cells. In the present study, we report that EBV-transformed lymphoblastoid B-cell lines (LCLs) and primary PTLDs overexpress galectin-1 (Gal1), a carbohydrate-binding lectin that induces tolerogenic dendritic cells and triggers the selective apoptosis of CD4+ Th1 and Th17 cells and cytotoxic T cells. In transcriptional reporter assays, LMP2A and LMP1 each increased Gal1-driven luciferase expression, and the combination of LMP2A and LMP1 was additive. In addition, small interfering RNA (siRNA)–mediated depletion of LMP2A decreased Gal1 protein abundance in EBV-transformed LCLs. Gal1 expression in LCLs was dependent on both activating protein 1 (AP-1) and PI3K. A newly developed neutralizing Gal1 mAb selectively inhibited Gal1-mediated apoptosis of EBV-specific CD8+ T cells. Given the tolerogenic and immunosuppressive function of Gal1, antibody-mediated Gal1 neutralization may represent a novel immunotherapeutic strategy for PTLD and other Gal1-expressing tumors.

A peptide derived from the Wiskott-Aldrich syndrome (WAS) protein-interacting protein (WIP) restores WAS protein level and actin cytoskeleton reorganization in lymphocytes from patients with WAS mutations that disrupt WIP binding

The Wiskott-Aldrich syndrome (WAS) and X-linked thrombocytopenia (XLT) are caused by mutations in WAS, which encodes for WAS protein (WASP). The WASP-interacting protein (WIP) stabilizes WASP, as evidenced by severely decreased WASP levels in T cells from WIP-deficient mice. The majority of missense mutations in patients with WAS/XLT are located in the WIP-binding domain of WASP and might result in dissociation of the WASP-WIP complex and WASP degradation. To restore WASP levels and correct T-cell function in WAS/XLT patients with mutations in the WIP-binding domain of WASP. WIP, and a WIP-derived 41-amino acid-long peptide, which interacts with WASP and was designated nanoWIP (nWIP), were fused to enhanced green fluorescent protein and introduced by electroporation into EBV-transformed B cells, and by retroviral transduction into purified blood T cells from patients with WAS. WASP levels were measured by intracellular fluorescence-activated cell sorting staining. The actin cytoskeleton was visualized by intracellular phalloidin staining. Introduction of WIP and nWIP restored WASP levels to normal in EBV-transformed B-cell lines from XLT patients with missense mutations in the WIP-binding domain of WASP and residual WASP levels, and corrected the defective spreading and pseudopodia formation of their T cells in response to immobilized anti-CD3. A WASP-binding WIP-derived peptide stabilizes WASP in cells from XLT patients with missense mutations that disrupt WIP binding, and corrects their T-cell actin cytoskeleton defect. This may provide a novel therapeutic strategy for these patients.

Expression and Targeted Inhibition of the Immunoregulatory Carbohydrate-Binding Lectin, Galectin 1, in EBV-Driven Post-Transplant Lymphoproliferative Disorders.

Abstract 96EBV-associated post-transplant lymphoproliferative disorders (PTLD) are potentially fatal EBV-driven B cell proliferations that develop in immunocompromised solid organ or bone marrow allograft recipients. Proliferation of EBV-infected B cells in PTLD is maintained by expression of EBV latent genes such as latent membrane protein 1 (LMP1) and LMP2A, viral immune evasion strategies, and impaired host immune surveillance. Management of early PTLD lesions is currently based on reduction or withdrawal of immunosuppression which increases the risk of graft rejection. For these reasons, alternative treatments that specifically boost host anti-EBV immune responses are needed.To identify novel T-cell inhibitory molecules induced by EBV and expressed in PTLDs, we compared the transcription profiles of a series of EBV-transformed lymphoblastoid B-cell lines (LCLs) and diffuse large B-cell lymphoma (DLBCL) cell lines. The LCLs overexpressed galectin-1 (Gal-1), a carbohydrate-binding lectin that induces tolerogenic dendritic cells and triggers the selective apoptosis of CD4+ Th1 and CD8+ cytotoxic T cells. Gal-1 protein expression was also uniformly high in LCLs and low or undetectable in DLBCL lines by western blotting. Immunohistochemical staining of primary tumors confirmed abundant Gal-1 expression in PTLD B-cells, whereas DLBCLs were negative.To elucidate the mechanisms responsible for Gal-1 overexpression in EBV-associated PTLD, we performed transcriptional reporter assays using cotransfected FLAG-tagged LMP1 and/or LMP2A and a luciferase reporter vector driven by Gal-1 promoter. LMP2A and LMP1 each increased Gal-1-driven luciferase expression 3- to 6-fold, respectively, and the combination of LMP2A and LMP1 was synergistic. In complementary experiments, siRNA-mediated depletion of LMP2A decreased Gal-1 protein abundance in EBV-transformed lymphoblastoid cells. The mechanism of LMP1 and LMP2A-induced Gal-1 expression in LCLs was both AP-1 and PI3K-dependent; the PI3K selective inhibitor, LY294002, markedly inhibited Gal-1 expression in these cells.Given the tolerogenic and immunosuppressive function of Gal-1 and the EBV-driven overexpression of the lectin in LCLs and primary PTLDs, we postulated that Gal-1 might be a targetable immune escape mechanism in EBV-associated malignancies. For these reasons, we assessed the neutralizing activity of a recently developed series of anti-Gal1 monoclonal antibodies. After confirming that these reagents selectively identified both recombinant and native Gal1, we asked whether certain Gal-1 monoclonal antibodies neutralized recombinant Gal-1-mediated apoptosis of EBV-specific polyclonal, largely CD8+, T-cells. Preincubation of human recombinant Gal-1 (hrGal-1) with Gal-1 monoclonal antibodies, but not with isotype-matched controls, selectively inhibited hrGal-1 induced apoptosis of EBV-specific CD8+ T-cells.Taken together, these studies indicate that EBV-transformed LCLs and primary EBV-associated PTLDs express Gal-1 in a LMP-1 and LMP2A-dependent manner. Given the acknowledged role of Gal-1 in mediating an immunopriviledged tumor microenvironment, the lectin likely inhibits the host anti-tumor response in these tumors. Antibody-mediated neutralization of secreted Gal-1 may represent a novel immunotherapeutic strategy in PTLD and other Gal1-expressing tumors. Disclosures:No relevant conflicts of interest to declare.

Epstein-Barr virus-transformation of B-cell lines in ovarian cancer patients: feasibility of genomic storage for unlimited use

The aim of the current study is to test whether immortalized B-lymphocyte cell line via Ebstein-Barr virus (EBV) transformation is feasible and can be an unlimited source of genome wide study. We obtained peripheral whole blood from 5 ovarian cancer patients and immortalized the B-cell lines using EBV transformation. The success rate was analyzed and the bio-identity of the genome was performed using human leukocyte antigen (HLA) identity test. EBV transformation was successful in all 5 cases (95% confidence interval, 46.3% to 100%). After cryopreservation of EBV-transformed B-cell lines and subsequent thawing, we observed that all cell lines were viable and proliferative. To check bio-identity, HLA-A, B, and DR were tested between the genome of the original samples and the transformed samples. The HLA typing revealed that all observed HLA-A, B, and DR type was identical in 5 cases before and after EBV-transformation. The current results suggest that EBV-transformation of peripheral blood is an efficient tool in genome banking. The EBV-transformed B-cell lines may be a valuable resource of genome in multi-center translational research by the Korean Gynecologic Oncology Group.

IL-15 Augments in-Vitro Expansion and Functional Activity of Antigen-Specific Effector Memory T-Cells (TEM) While Co-Expression of IL-15 and IL-15 Rα on Antigen Presenting Cells Also Promotes Expansion of Central Memory T-Cells (TCM)

Adoptive immunotherapy with in-vitro expanded antigen-specific T cells (TC) is often hampered by the extended culture times required to generate sufficient numbers of antigen- specific TC and their limited persistence in-vivo. IL-2 predominantly supports the generation of short-lived effector memory (TEM) and effector (TE) CD8+ TC without expanding the CD62L+ and CCR7+ central memory T cells (TCM), which may persist for long periods in vivo. We examined the potential of IL-15 to foster growth of CMVpp65- specific TC as well as expansion of both TEM and TCM CD8+ TC.Accordingly, TC from 3 seropositive donors bearing HLA A0201 were sensitized in-vitro using NIH 3T3 based artificial antigen presenting cells transduced to express B7.1, ICAM-1, LFA-3, β2-M and HLA A0201 heavy chain as well as CMVpp65 (A2-AAPC) in presence of exogenous IL-2, IL-15 or IL-15 plus IL-2. To assess if trans-presentation of IL-15 by the receptor alpha on AAPCs leads to enhancement of IL-15 activity, we sequentially transduced A2-AAPCs with the IL-15 and IL-15 receptor alpha cDNA (A2- AAPC IL-15/IL-15Rα). TC were then cultured using A2-AAPC with eitherIL-2 (20U/ml)IL-15 (10ng/ml)IL-2 plus IL-15 or withA2-AAPC IL-15/IL-15Rα orA2-AAPC IL-15Rα + IL-2 (20U/ml).TC sensitized using either A2-AAPC IL-15/IL-15Rα or A2-AAPC + IL-15 demonstrated ~ 1500–2000 fold expansion of CMVpp65 A2-NLV tetramer (+) CD8+ TC, compared to a 300–600 fold expansion using A2-AAPC + IL-2 after 21–28 days in culture. Cultures containing IL-15 generated 25–70 ×106 A2-NLV tetramer (+) CD8+ TC in comparison to 10–18 ×106 in cultures with IL-2. At 7–10 days, ~20% of the tetramer (+) TC demonstrated a TCM phenotype (CD62L+ and CCR7+) in all cultures, with 75% TEM and 5% TE. By 21- 28 days, no TCM were detected among tetramer (+) TC in cultures containing exogenous IL-2, IL-15 or IL-2 plus IL-15. However, TC sensitized with A2-AAPC IL-15/IL-15Rα still contained 10–12% (~7×106) tetramer (+) CD8+ TCM which further increased through day 35. In functional assays, TC sensitized in the presence of IL-15 or AAPCs expressing IL-15 and IL-15Rα elicited superior CMV-specific responses with 7–20% CD8+ TC demonstrating CMVpp65 epitope specific interferon gamma production compared to 2–3% in cultures with IL-2. Cytotoxic activity against CMV pp65 peptide loaded autologous EBV transformed B-cell lines (E:T= 10:1) was higher for TC sensitized with IL-15 (80%) compared to cultures with IL-2 (60%). The cytotoxic activity against HLA mismatched targets or K562 was <5% in all cultures.In conclusion, our data demonstrate higher yields and augmented function in antigen specific T cells cultured with IL-15. TC sensitized with A2-AAPC-CMVpp65 together with IL-15 +/− IL-2 only supported sustained expansion of TEM and TE. In contrast, TC sensitized with A2-AAPC IL-15/IL-15Rα also supported sustained expansion of TCM.

A New Human CD20 Spliced mRNA as a Potential Molecular Marker for the Follow-up of B-Cell Malignancies.

The human CD20 molecule (hCD20) is a B-cell lineage specific marker expressed by normal and leukemic B cells from the pre-B to the plasma-cell stages and a target for Rituximab (Rx) immunotherapy. During the course of a CD20 RTPCR on B-cell line cDNA, we unexpectedly obtained a shorter PCR product (ΔCD20) in addition to the expected 894bp PCR product (wtCD20). Sequencing analysis revealed that this additional fragment was identical to Genbank published CD20 sequence, but lacked 501bp. In silico analysis of the wtCD20 sequence, using Genesplicer and NetGene 2 softwares, showed donor and acceptor sites (nt112 & nt611 respectively, from ATG codon) matching the newly identified spliced DCD20 form and a branched site located in nt595 exon 3 of the gene respectively. The CD20 spliced mRNA form links part of the end of exon 3 to that of exon 7. The truncated sequence is on the reading frame and can code a putative protein of 130 amino-acids (~15KD) including the intracellular C- terminal domain with part of the transmembrane 1 (TM1) domain and the end of the N-terminal intracellular domain. The extracellular domain and large parts of the 4 TM segments are deleted suggesting that the putative ΔCD20 may be a non-anchored membrane protein. Using CD20 PCR assays amplifying either both wtCD20 and ΔCD20 forms or ΔCD20 alone, we detected the truncated mRNA DCD20 form in different B cell lines (n=12) but not in different T-cell lines (n=4). With a QPCR assay allowing for the specific quantification of either wtCD20 or truncated DCD20 mRNA we also detected the ΔCD20 spliced form [expressed as relative % of ΔCD20: R = (ΔCD20/wtCD20+ΔCD20) × 100] in in-vitro EBV-transformed B-cell lines (2.9 ± 4.51%, n=6); as well as in CD19+ cell sorted cells from tonsillectomy samples (9 ± 2.2%, n=7) and in-vitro B blast cells (14 ± 7.8%, n=5). Interestingly, screening of a panel of B-cell hematologic malignancies showed that the spliced form is detectable at various levels. We found a mean of 3.6 ± 5.1% in B-ALL (n=27); 3.9 ± 5.3% in follicular lymphomas (n=5); 2.9 ± 4.5% in mantle lymphomas (n=6); 3.2 ± 2.2% in high grade lymphomas (n=5); and 0.1 ± 0.2% in B-CLL (n=8). However, this spliced form was not detected in peripheral blood mononuclear cells or in immunomagnetically-sorted CD19+ or CD20+ blood cells from healthy donors. To explore clinical relevance, molecular monitoring of ΔCD20 in the marrow was performed in 2 patients. A 1rst patient with mantle lymphoma, showed an R of 4.2% at diagnosis (diag). After chemotherapy (VAD + chloraminophen) + Rx, R was 2.2% at +12 mo (×1.9 decrease from diag) and 1.8% at +18mo (×1.3 decrease/+12mo). This was in accordance with the cytological (absence of lymphoma cells) and the molecular response (absence of cyclinD1 overexpression). A second patient, with a Phi+ B-ALL, showed an ×13.7 R-decrease compared to diag (from 4.1% to 0.3%) following treatment according to the molecular response. Interestingly, R increased up to 2.6% at +8 months (×8.6/6mo) and 3% (×1.1/8mo) at +12 months after treatment, while molecular and cytological relapse was evidenced only 12 mo after treatment. In conclusion, we report evidence of a novel alternatively spliced transcript of the hCD20 gene, specifically expressed at detectable levels in leukemic, lymphoma, activated or EBV-transformed B cells, but not in normal resting B cells. Further experiments will determine whether the ΔCD20 mRNA spliced form modulates wtCD20 expression and thus influences the response to Rx treatment in hematologic B diseases. However, our initial results suggest that DCD20 quantification may be an indicator of minimal residual disease, as a potential predictive marker of relapse, especially in patients with no other molecular marker.

Cyclosporin A Up-Regulates and Activates Protein Kinase C-ζ in EBV-Infected and EBV-Transformed Human B-Cells

Protein Kinase C (PKC) is a family of enzymes that plays a key role in cell signaling pathways leading to cellular activation and proliferation. Conventional PKC (cPKC) is dependent on calcium for activation. We have proposed that cyclosporin A (CsA), despite being a calcineurin inhibitor, will activate PKC in B cells, thus promoting Epstein-Barr virus (EBV)-induced transformation. Here we show that CsA promoted atypical PKC isoform PKC-zeta in B cells. Western-blot was used to assay PKC-zeta protein level in EBV-B cells. Confocal microscopy was used to assay PKC-zeta translocation from cytosol to cell membrane, a known process of PKC activation. CsA (500 ng/mL) time dependently increased PKC-zeta from control of 7055 units to 7145, 10,805, 10,914, and 12,705 units, respectively, after 15 min, 1 h, 12 h, and 24 h of incubation in EBV-transformed human B-cell line (LCL). CsA increased PKC-zeta expression was inhibited 50% by Vit.E (40 microM) indicating that this effect may be due to oxidative stress induced by CsA. Indeed, after oxidant H(2)O(2) (0.1 mM) treatment, PKC-zeta protein level in LCL cells increased 124%, 257%, 349%, and 359% after 15 min, 1 h, 12 h, and 24 h of culture compared with control. Addition of Vit.E (40 microM) in H(2)O(2) (0.1 mM) treatment and then with Vit.E in the culture decreased PKC-zeta level in LCL cells 26%, 20%, 41%, and 60% after 15 min, 1 h, 12 h, and 24 h of culture. In confocal microscopy in Jurkat T cell line, phorbol 12-myristate 13-acetate (PMA) activated cPKC isoform PKCalpha after 30 min treatment and activated PKC-zeta after 60 min treatment. CsA inhibited PMA activation of PKC-alpha, but not PKC-zeta. CsA alone did not activate PKC-alpha or PKC-zeta in Jurkat T cells. In LCL and in EBV-infected human B-cells, PMA stimulated PKC-alpha activation after 30 min treatment and stimulated PKC-zeta activation after 60 min treatment. CsA inhibited PMA activation of PKC-alpha, but not PKC-zeta. In addition, CsA activated PKC-zeta in the EBV-transformed and EBV-infected human B cells. These experiments show that CsA-induced oxidative stress caused PKC-zeta up-regulation in LCL cells, and show the differential effect of CsA in the PKC signaling pathways in T cells versus B cells. CsA-induced PKC-zeta activation may be an important signaling step in EBV-induced post-transplant lymphoproliferative disorders.