EBV Cell Lines Research Articles

Abstract 3788: Sensitive detection of oncoviruses integrated into a comprehensive tumor immuno-genomics platform

Abstract Introduction HPV, HBV, HCV and EBV viruses are causally linked to over 11% of cancers worldwide while KSHV, HTLV and MCV are linked to an additional 1%. As use of immunotherapy expands to a broader variety of cancers, it is important to understand how these oncoviruses may be impacting the overall immune response in patients as part of the tumor and tumor microenvironment. However, typical cancer diagnostic and genomic biomarker assays do not include oncovirus detection. Methods To enable detection of these oncoviruses, we have developed ImmunoID NeXT, a novel augmented exome and transcriptome based platform that comprehensively characterizes tumor and tumor microenvironment from a single sample. As part of the platform assay design we developed targeting for the following oncoviruses: HPV, HBV, HCV, EBV, KSHV and HTLV. Furthermore we developed analytics for detecting these viruses from both the DNA and RNA data. To test the ability of the platform to detect these oncoviruses, we identified a set of 11 EBV cell lines from Coriell in which EBV was used as a transformant. We obtained another 23 cell lines from ATCC containing HPV16, HPV18, HPV45, HPV68, HBV, EBV, KSHV, HTLV1 and HTLV2 in which the oncoviruses were known to be in the tumors from which the cell lines were created. We have also started running a series a FFPE tumor samples of unknown status. We extracted DNA and RNA from all these samples, made libraries and sequenced them on a NovaSeq to 30G. High quality read counts were computed for all targeted oncoviruses. Results We detected EBV in all the Coriell cell lines in both DNA and RNA indicating strong sensitivity of the platform. Wide dynamic range suggests quantification may be possible as well. In the DNA there was no signal from any other oncovirus indicating high specificity. In the ATCC samples we detected 23 out of 23 oncoviruses expected in both the DNA and RNA. We detected all the different types of oncoviruses that we targeted except for HCV because it wasn’t in any sample. In all but one case the signals were strong. False positive detections appear to be rare. Much less than 1% of the overall exome/transcriptome reads map to oncoviruses in all cases. Conclusions Oncovirus detection integrated into an augmented exome and transcriptome assay is sensitive and specific. Oncoviral characterization of the tumor as part of a comprehensive tumor immunogenomics platform can serve and an important additional biomarker for understanding immunotherapy response. Citation Format: Gabor Bartha, Robin Li, Shujun Luo, John West, Richard Chen. Sensitive detection of oncoviruses integrated into a comprehensive tumor immuno-genomics platform [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3788.

Evaluation of relative quantification of alternatively spliced transcripts using droplet digital PCR

IntroductionFor the relative quantification of isoform expression, RT-qPCR has been the gold standard for over a decade. More recently, digital PCR is becoming widely implemented, as it is promised to be more accurate, sensitive and less affected by inhibitors, without the need for standard curves. In this study we evaluated RT-qPCR versus RT-droplet digital PCR (ddPCR) for the relative quantification of isoforms in controls and carriers of the splice site mutation BRCA1 c.212+3A>G, associated with increased expression of several isoforms. Materials and methodsRNA was extracted from EBV cell lines of controls and heterozygous BRCA1 c.212+3A>G carriers. Transcript-specific plasmids were available to determine the efficiency, precision, reproducibility and accuracy of each method. ResultsBoth ddPCR and RT-qPCR were able to accurately quantify all targets and showed the same LOB, LOD and LOQ; also precision and reproducibility were similar. Both techniques have the same dynamic range and linearity at biologically relevant template concentrations. However, a significantly higher cost and workload was required for ddPCR experiments. ConclusionsOur study recognizes the potential and validity of digital PCR but shows the value of a highly optimized qPCR for the relative quantification of isoforms. Cost efficiency and simplicity turned out to be better for RT-qPCR.

OR42: RNA AND PROTEIN EXPRESSION OF HLA-A∗23:19Q

It is clinically relevant to assign null alleles since they affect the donor selection in stem cell transplantation due to their absence on the cell surface. Questionable alleles however, are a problem since their expression profile is unknown, which makes it unclear how they should be considered in matching criteria. The nomenclature committee assigns alleles as questionable when they carry a mutation that has previously been shown to have a significant effect on cell surface expression but has not been confirmed. In this study the expression profile of one of these questionable alleles HLA-A∗23:19Q is addressed. This allele has one known substitution in exon 3 at position 619 (G > A) compared to HLA-A∗23:01:01. We encountered HLA-A∗23:19Q in a sample of our population study of Guadeloupe (HLA-A∗23:19Q, 29:02:01). Serological typing of the sample showed only the presence of A29, suggesting that A∗23:19Q was not expressed on the cell surface, whereas the presence of the A∗23:19Q allele was confirmed by SBT DNA sequencing. Interestingly, allele specific sequencing of mRNA revealed the absence of an intact full-length ‘normal’ mRNA product and the presence of two alternatively spliced mRNAs. Sequencing these two variants resulted in one variant missing exon 3 and the other variant retaining introns 2 and 3. Both these variants will not encode a regular A23 molecule because of the lack of the α 2 domain, whereas intron 2 introduces a premature stop codon. To confirm the lack of expression of A∗23:19Q the patient sample was transformed into an EBV cell line and used for flow cytometry analysis of HLA cell surface expression. The absence of HLA-A∗23:19Q on the cell surface was shown by using a directly labelled A9 monoclonal antibody and indirect labelling of multiple patient sera having polyclonal antibodies directed against A9 and A23. Based upon the lack of detection of A∗23:19Q cell surface expression and the absence of a full length mRNA transcript, this study indicates that A∗23:19Q should be reclassified as a null allele.

The Signal Peptide of the Tumor-shared Antigen Midkine Hosts CD4+ T Cell Epitopes

The CD4 T cell response to the tumor antigen Midkine was unknown. Most of the T cell response to Midkine relies on T cell epitopes contained in its signal peptide. The signal peptide of Midkine is accessible to HLA class II pathway for CD4 T cell presentation. It is a new function for signal peptides to contribute to tumor-specific CD4 T cell response. Because of the key role of CD4 T cell response in immunity to tumors, we investigated the CD4(+) T cell response to the recently identified tumor antigen Midkine (MDK). By weekly stimulations of T lymphocytes harvested from seven HLA-DR-typed healthy donors, we derived CD4(+) T cell lines specific for eight MDK peptides. Most of the T cell lines reacted with the peptides 9-23 and 14-28, located in and overlapping the MDK signal peptide, respectively. Accordingly, the MDK signal peptide appeared to be rich in good binders to common HLA-DR molecules. The peptide 9-23-specific T cell lines were specifically stimulated by autologous dendritic cells loaded with lysates of MDK-transfected cells or with lysates of tumor cells naturally expressing the MDK protein. One T cell line was stimulated by HLA-compatible MDK-transfected tumor cells. By contrast, the peptide 14-28-specific T cell lines were not stimulated in any of these conditions. Our data demonstrate that CD4(+) T cell epitopes present in the signal peptide can be accessible to recognition by CD4(+) T cells and may therefore contribute to tumor immunity, whereas a peptide overlapping the junction between the signal peptide and the mature protein is not.

A microdeletion proximal of the critical deletion region is associated with mild Wolf–Hirschhorn syndrome

It is generally accepted that the facial phenotype of Wolf-Hirschhorn syndrome is caused by deletions of either Wolf-Hirschhorn critical regions 1 or 2 (WHSCR 1-2). Here, we identify a 432 kb deletion located 600 kb proximal to both WHSCR1-2 in a patient with a WHS facial phenotype. Seven genes are underlying this deletion region including FAM193a, ADD1, NOP14, GRK4, MFSD10, SH3BP2, TNIP2. The clinical diagnosis of WHS facial phenotype was confirmed by 3D facial analysis using dense surface modeling. Our results suggest that the WHSCR1-2 flanking sequence contributes directly or indirectly to the severity of WHS. Sequencing the Wolf-Hirschhorn syndrome candidate 1 and 2 genes did not reveal any mutations. Long range position effects of the deletion that could influence gene expression within the WHSCR were excluded in EBV cell lines derived from patient lymphoblasts. We hypothesize that either (1) this locus harbors regulatory sequences which affect gene expression in the WHSCR1-2 in a defined temporal and spatial developmental window or (2) that this locus is additive to deletions of WHSCR1-2 increasing the phenotypic expression.

The kConFab experience – 14 years of biobanking

kConFab, the Australian/New Zealand consortium for research into families at high risk of breast and ovarian cancer, has completed collection & recruitment of 1,421 families during the past 14 years. Biological material, genetic, epidemiological, and psychosocial data are collected from affected and unaffected, female and male participants over the age of 18. This material is available to peer reviewed, ethically approved and funded research projects. At present, kConFab supplies material to 52 research projects world-wide. The kConFab biological repository contains blood specimens from a total of 12,340 participants and 233 best friend controls. The standardized blood processing protocol produces plasma, non lymph, blood pellet and white blood cell fractions. White blood cells undergo EBV transformation which can be used by in functional assays or as a replacement source of DNA/RNA. To date, 1511 unique EBV cell line transformations are available. As of July 2011, 97% of kConFab families have had genetic testing; identifying 35% of families with a pathogenic, large genomic rearrangement (LGR) or splice site mutation in either BRCA1 or BRCA2. An additional 11% of families carry unclassified variants in BRCA1 or BRCA2; with a further 0.7 % with mutations in the ATM, CHEK2 or TP53 genes. Of the 1,924 female participants who harbour the germline mutation, 66% are affected with breast or ovarian cancer. kConFab has collected a total of 898 fresh tissue collections, including prophylactic mastectomy and oophorectomy specimens; and has a large collection of archival specimens. The tissue bank consists primarily of breast and ovarian tissue (tumour and normal), with a small proportion of other tissues. Tumour tissue comprises 27% of the bank. Following collection, a full research pathology review is conducted, wherein features such percentage tumour, normal epithelial, lymph and necrotic components are scored. kConFab has constructed a total of 23 tissue microarrays (TMAs) (both sporadic and familial tumours) from our tissue bio bank. Where possible, tumour is matched to normal from the same archival block. kConFab are currently working to supplement our glass slide archive with a digital slide repository. This will provide researchers with high resolution, high quality whole slide digital images for ease of transport, storage, review and analysis. Currently we have >1000 slides scanned for more than 600 participants. The kConFab resource enables researchers to answer important questions relating to familial aspects of breast cancer. Information about the kConFab resource and the application process is available on the web site (http://www.kconfab.org).

Reprogramming of EBV-immortalized B-lymphocyte cell lines into induced pluripotent stem cells

AbstractEBV-immortalized B lymphocyte cell lines have been widely banked for studying a variety of diseases, including rare genetic disorders. These cell lines represent an important resource for disease modeling with the induced pluripotent stem cell (iPSC) technology. Here we report the generation of iPSCs from EBV-immortalized B-cell lines derived from multiple inherited disease patients via a nonviral method. The reprogramming method for the EBV cell lines involves a distinct protocol compared with that of patient fibroblasts. The B-cell line–derived iPSCs expressed pluripotency markers, retained the inherited mutation and the parental V(D)J rearrangement profile, and differentiated into all 3 germ layer cell types. There was no integration of the reprogramming-related transgenes or the EBV-associated genes in these iPSCs. The ability to reprogram the widely banked patient B-cell lines will offer an unprecedented opportunity to generate human disease models and provide novel drug therapies.

Report on the 14th international society of blood transfusion platelet immunology workshop

The aims of the 14th ISBT Platelet Immunology Workshop were to evaluate in-house methods for detection of antibodies to human platelet antigens, to compare the sensitivity and specificity of antibody detection using a panel of monoclonal antibodies and to evaluate genotyping methods and establish procedures for drug-dependent antibody detection. Forty-two laboratories from 23 countries participated. Samples and reagents provided for the five different exercises. The ability of participating laboratories to correctly identify the HPA antibody specificity in the nine samples ranged from 20% to 97%. The greatest difficulty was observed with samples that contained antibodies against HPA-3b and GPIV. The significant differences in optical density values by monoclonal antibody of immobilization of platelet antigens (MAIPA) assay were observed when testing the same platelet-specific antibodies. HPA genotyping of DNA with novel mutations did not significantly affect the results. The overall average discrepancy rate was 2·15% for genotyping of 10 DNA samples from well-characterized Epstein–Barr virus transformed cell lines. For detection of drug-dependent antibodies, excellent results for specificity and sensitivity were obtained by the laboratories using the MAIPA and flow cytometry. Most laboratories were able to identify the majority of HPA antibodies; however, significant disparities were observed in proficiency testing. MAIPA assay sensitivity is influenced by the monoclonal antibody clone used. DNA with new mutations and EBV cell lines are valuable samples to ensure accurate genotyping. A sensitive and specific drug-dependent antibody assay performed well in the hands of participants.

A Large Ribosomal Subunit Protein Abnormality in Diamond-Blackfan Anemia (DBA).

DBA is an inherited bone marrow failure syndrome characterized by hypoproliferative anemia, congenital abnormalities and cancer predisposition. Ribosomal genes RPS19 and 24 are mutated in 25% and 3% of DBA patients respectively. To identify additional genetic abnormalities in DBA, we evaluated 2 unrelated children with DBA and sub-telomeric deletions of chromosome 3q by comparative genomic hybridization. The larger deletion spanned 11 Mb from 3q28 to the telomeric region and included 72 gene candidates. The second deletion involved 4 Mb from 3q29 to the telomeric end and included 52 known or hypothetical genes. The overlapping deletion region contained a previously reported 1.5 Mb microdeletion-associated syndrome that did not involve hematologic abnormalities, leaving 24 candidate genes. Gene expression microarray analysis from patient-derived EBV cell lines demonstrated down regulation of 7 of these candidate genes, one of which was RPL35a, a component of the large ribosomal subunit. We screened for mutations of RPL35a by direct sequencing of PCR-amplified genomic DNA from 149 DBA probands (125 sporadic, 24 familial) and 180 normal control subjects. We identified three probands with sequence changes in the RPL35a coding region: 1) an in-frame deletion in exon 3 (82-84CTT), causing a deletion of leucine at codon 28, 2) a nonsense mutation in exon 4 (298C>T), leading to an Arg102Stop and a 9 amino acid C-terminal truncation and 3) a missense mutation in exon 3 (97G>A) leading to a Val33Ile change. In the patient derived EBV cell line, the latter sequence change also resulted in an aberrant exon 3 splice site leading to a frame shift following codon 32. All of the probands with RPL35a mutations were sporadic cases. These sequence variations were not observed in the control subjects. Four lentiviral-based siRNA constructs targeting RPL35a were used to test the functional consequences of reduced RPL35a expression. Hematopoietic cell lines (TF-1 and UT-7/epo) transduced with the RPL35a directed siRNA constructs demonstrated decreased growth and viability compared to control siRNAs. Northern blot analysis demonstrated abnormal processing of large ribosomal subunit RNA with decreased mature 5.8S and 28S as well as decreased precursor 12S and 32S rRNA. Orthophosphate labeling confirmed a kinetic defect in large subunit rRNA processing, characterized by increased amounts of 45S and 41S rRNA with decreases of the precursors to and the mature 28S and 5.8S rRNAs. Mature 18S rRNA levels were unaffected, suggesting a defect in rRNA processing within the first internal transcribed sequence (ITS1). These data demonstrate that DBA can be caused by alterations in large as well as small ribosomal subunit proteins. These observations further support the hypothesis that altered ribosome homeostasis and function, rather than extra-ribosomal gene functions, is the central mechanism leading to DBA.

B7-1 and 4-1BB ligand expression on a myeloma cell line makes it possible to expand autologous tumor-specific cytotoxic T cells in vitro

The aim of this study was to confer an antigen-presenting cell (APC) ability on multiple myeloma cell lines (HMCLs) using B7-1 and/or 4-1BBL gene transfer. HMCLs were retrovirally transduced with B7-1 and/or 4-1BBL cDNAs. Allogeneic or autologous T cells were stimulated by coculture with B7-1- and/or 4-1BBL-transduced HMCLs in the presence of interleukin-2. T cell clones were obtained by limiting dilution. T-cell activation was assessed by interferon-gamma Elispot assays and cytotoxicity by (51)Cr release assays. Neither primary multiple myeloma cells (MMCs) nor HMCLs expressed B7-1 or 4-1BBL, and these molecules could not be induced by CD40 triggering. HMCLs failed to stimulate allogeneic or autologous T cells. Transduction of HMCLs with B7-1 and/or 4-1BBL retroviruses induced a high expression of B7-1 and 4-1BBL molecules and a strong T-cell activation ability. Long-term cultured CD8(+) T-cell lines could be obtained by stimulation with the autologous B7-1/4-1BBL XG-19 HMCL. These cytotoxic T lymphocytes (CTL) efficiently killed the autologous parental XG-19 HMCL as well as autologous primary MMCs and allogeneic HMCLs. They did not kill autologous CD34 cells and autologous EBV cell line or natural killer target K562 cells. Cloned CTL could recognize allogeneic HMCLs, demonstrating that a shared anti-MMC repertoire was expanded. Transduction with B7-1 and 4-1BBL retroviruses turned HMCLs into efficient APCs. It permitted the long-term expansion of autologous anti-tumor CTL with a shared anti-MMC repertoire, for one HMCL. These data suggest developing an immunotherapy using modified tumor cells in patients with multiple myeloma.

Canonical NF-kB Pathway Is Not Constitutively Activated and May Not Be the Major Bortezomib Target in Mantle Cell Lymphoma (MCL).

Bortezomib, a proteasome inhibitor, has promising activity in MCL. Bortezomib mechanism of action is complex since multiple proteins and cellular pathways are regulated by proteasome degradation. By blocking the degradation of IKBa, the canonical NF-kB pathway is one of the major Bortezomib target in lymphoid malignancies such as multiple myeloma. NF-kB activation has not been extensively assessed in other lymphoid malignancies such as MCL. NF-kB complexes (heterodimers P50/P65) were assessed by EMSA assays in REC GRANTA 519, NCEB, JEKO, JUN and UPN1 MCL cell-lines. EBV was present in 3/6 cell lines. P50/P65 complexes were detected in all EBV positive cell lines and only in 1/3 EBV negative cell-lines (JEKO). No nuclear P65 protein was detected by immunofluorescence or Western-blot analysis in the 2 other negative EBV cell lines (REC and UPN1). NF-kB transcriptional activity was measured by a luciferase-based reporter gene assay. Spontaneous NF-kB activity was low in UPN1, REC and JEKO compared to GRANTA 519 EBV positive cell-line (3 to 4 time lower) but can be strongly activated (up to 10 fold) using MEKK cotransfection assay. This suggest that NF-kB is functional, but not constitutively activated. Stable infections with Migr1-IRES-GFP IkBM (dominant negative IKB) or empty vector, were performed in GRANTA 519 EBV positive as well as in UPN1 cell line. No differences in proliferation or apoptosis were observed in UPN1 stably infected with IkBM or with the empty vector but GRANTA 519 EBV positive showed increased apoptosis and proliferation inhibition when infected with IkBM. Likewise, no P50/P65 heterodimers complexes were detected by EMSA in 4 patients with MCL. In vitro assays showed that UPN1 and JEKO cell lines had comparable sensitivities to Bortezomib than Multiple myeloma cell lines reported in the literature (IC 50: 6 nM and 12 nM respectively). This was also true for the 3 patients lymphoma cells assessed in vitro (12 nM). Taken together these results suggest that canonical NF-kB activation pathway is not constitutively active in EBV negative MCL cell lines and patients samples and could be associated with EBV infection in some MCL cell-lines. Therefore, this strongly suggests that Bortezomib target other molecules in MCL.

Validation of Flow Cytometric Measurements of NK Cytotoxicity Against Chromium Release Assay for Use in Clinical Monitoring of Immune Function.

Chromium (51Cr) release assays have long been a standard technique for measuring the cytolytic activity of various immune effector cells. Although relatively easy to perform, these assays suffer from problems with sensitivity and require special radiation precautions limiting their routine use in clinical laboratories. Several flow cytometric techniques measuring cytotoxicity have been introduced and used as a measure of immune response. These assays are more rapid than the 51Cr based assay and have the added advantage of freeing the laboratory from the precautions required when working with radioactivity. Such testing has however largely been restricted to the research setting. In an attempt to extend these benefits to a clinical laboratory, we attempted to validate two commercially available flow cytometry kits (CyToxiLux Plus® & GranToxiLux®, OncoImmunin, Gathersburg, MD) measuring either caspase or granzyme B activity inside K562 target cells induced by interaction with NK cells. These assays were then compared with a standard NK cytotoxicity assay using 51Cr release technique. With K562 as a target, we parallel tested three CD56+CD3-KIR- NK clones; both the 51Cr and caspase based cytotoxicity values were highly positive. With WH autologous EBV cell line as target, we parallel tested five NK clones and five normal control donors as effectors. As expected, all cytotoxicity values for both assays were <10%. The caspase and granzyme assays were then compared and the degree of separation in the flow cytometric plots of the granzyme based assay were found to be marginally superior facilitating analysis of the data. The granzyme B assay therefore was chosen for investigation in the clinical samples. The granzyme and 51Cr assays were then compared both in 49 normal control donors and in 10 patients. There was a high level of concordance between the two assays. Comparing all results using linear correlation yielded a squared correlation (R2) value of 0.6878. However, given the wide range of NK activity in normal control donors the degree of positivity may not be clinically significant. Therefore, the data was examined in the context of a positive 51Cr response. Using a 2×2 table to exam the level of agreement, the kappa statistic was 0.63, indicating good agreement between the assays. When progressively fewer effector cells were added to a fixed number of target cells, the % cytotoxicity decreased from 39% to 0% with the flow cytometric assay and from 26% to 1% with the 51Cr assay. In the normal control donors the positive values ranged from 17% to 65% cytotoxicity using the flow assay compared with the previous range of 15% to 72% established with 51Cr. Patients having cytotoxicity values below 17% were deemed as having an unreactive or abnormal low result. One of the patients with few CD56+CD3- NK cells had consistently low cytotoxicity each time tested. For the patients who have either undergone allogeneic stem cell transplantation or have a known immunodeficiency, further clinical follow-up will serve to establish the clinical relevance of these measurements. These preliminary data suggest that the flow cytometric technique can be used in the clinical setting for immune monitoring.

O.118 Management of patients with bisphosphonates-induced osteonecrosis

For the relative quantification of isoform expression, RT-qPCR has been the gold standard for over a decade. More recently, digital PCR is becoming widely implemented, as it is promised to be more accurate, sensitive and less affected by inhibitors, without the need for standard curves. In this study we evaluated RT-qPCR versus RT-droplet digital PCR (ddPCR) for the relative quantification of isoforms in controls and carriers of the splice site mutation BRCA1 c.212+3A>G, associated with increased expression of several isoforms.RNA was extracted from EBV cell lines of controls and heterozygous BRCA1 c.212+3A>G carriers. Transcript-specific plasmids were available to determine the efficiency, precision, reproducibility and accuracy of each method.Both ddPCR and RT-qPCR were able to accurately quantify all targets and showed the same LOB, LOD and LOQ; also precision and reproducibility were similar. Both techniques have the same dynamic range and linearity at biologically relevant template concentrations. However, a significantly higher cost and workload was required for ddPCR experiments.Our study recognizes the potential and validity of digital PCR but shows the value of a highly optimized qPCR for the relative quantification of isoforms. Cost efficiency and simplicity turned out to be better for RT-qPCR.

RAD001 (everolimus) down-regulates cyclin D3 and c-Myc and is particularly effective in the treatment of aggressive B-cell lymphomas

17573 Background: Aggressive B-cell lymphoma is recently found to be associated with constitutive activation of the PI3K/Akt pathway, and thereby is relatively resistant to apoptosis. Furthermore, activation of the PI3K/Akt signaling pathway can result in the upregulation of cyclin D3 and c-Myc through inhibition of the cap-independent RNA translation. Since mTOR is closely associated with the regulation of the translation process, and is a downstream mediator of the PI3K/Akt signalling pathway, this study aimed to examine if RAD001, an inhibitor of mTOR, can be effective in the treatment of aggressive B-cell lymphoma. Methods: Pfeiffer (diffuse large cell lymphoma), Ramos (EBV (−) Burkitt’s lymphoma), Raji (EBV (+) Burkitt’s lymphoma), and MC116 (EBV (−) undifferentiated lymphoma) cell lines were used in this study. Expression of pAkt, p70S6 kinase, pp70 S6 kinase, 4E-BP1, p4E-BP1, cyclin D3, and c-Myc was measured by immunoblotting. Results: Akt was constitutively activated in all four lymphoma cell lines. Downstream effectors of PI3/Akt signaling pathway, including p70S6 kinase and 4E-BP1, were also phosphorylated in these lymphoma cell lines. RAD001 downregulated p70S6 kinase and 4E-BP1, and the IC50s of growth suppression ranged from 5 to 50 nM, without significant difference between EBV (+) and EBV (−) cell lines. The IC50s of these lymphoma cell lines appeared to be much lower than those obtained from the carcinoma cell lines, suggesting that lymphomas may be particularly sensitive to growth inhibition by RAD001. RAD001 blocked cell cycle progression in G0/G1 phase in all four lymphoma cell lines while there was no significant increase in sub-G1 phases, suggesting that apoptosis was not the major mechanism of RAD001-induced growth inhibition. In addition, in parallel with the RAD001-induced growth inhibition, a dose-dependent down-regulation of the expression of cyclin D3 and c-Myc was demonstrated. Conclusions: The mechanism of action is at least partly due to downregulation of cyclin D3 and c-Myc, and subsequent G0/G1 block. Since overexpression of cyclin D3 and c-Myc is also closely associated with chemoresistance of aggressive B-cell lymphoma, RAD001 may be a useful adjunct in the treatment of this group of tumors. No significant financial relationships to disclose.

Donor T lymphocyte infusion following ex vivo depletion of donor anti-host reactivity by a specific anti–interleukin-2 receptor P55 chain immunotoxin

ALLOGENEIC stem cell transplantation is the treatment of choice for many hematologic malignancies or inherited disorders. In the partially incompatible HLA setting ex vivo T-cell depletion of the graft and posttransplantation immunosuppression prevent development of graft-versus-host disease (GVHD), but lead in turn to a delay in immune reconstitution and a concordant increase in the incidence of opportunistic infections and disease relapses. To deplete the T cells responsible for GVHD but retain T cells responsible for the anti-leukemic and antiinfectious activities, we have developed an in vitro procedure to allo-activate donor T cells responsible for GVHD and eliminate them with an immunotoxin that is reactive with the cell surface activation antigen CD25. We infused the remaining allodepleted T cells between day 15 and 47 into pediatric patients with acquired or congenital hematopoietic disorders who received hematopoietic stem cell transplants (HSCT) on day 0. We evaluated the rates of GVHD and opportunistic infections as well as the time to immune reconstitution. Sixteen infusions of allodepleted T cells, ranging from 1 to 8 10 cells/kg were administered to pediatric patients between March 1999 and April 2001, none of whom received prophylactic therapy for GVHD. Despite the risk of transfusing donor T cells activated against recipient alloantigens ex vivo, GVHD greater than grade II was not observed. Two patients (13%) developed grade 1 aGVHD limited to skin, and two others (13%) developed grade II aGVHD with one case involving both the skin and gastrointestinal tract. All cases resolved with treatment. Interestingly, all cases correlated with a residual alloproliferation of 1% (P .001) (data not shown). Three patients displayed an active cytomegalovirus (CMV) infection at the time of HSCT that required antiCMV treatment. In addition, one patient (#5) experienced pulmonary and cerebral EBV-induced lymphoproliferation and a varicella zoster virus infection. After the infusion of allodepleted T cells, two types of situations were observed. Patients who did not show major infectious complications or GVHD displayed a progressive increase in the CD4 T-cell compartment, particularly of naive CD45RA phenotype cells, which were detectable by week 14. In contrast, patients who were infected at time of T-cell infusion demonstrated a rapid expansion in the CD4 T-cell compartment with clinically significant numbers of cells, all expressing CD45RO by week 8. In an attempt to assess the ability of an allodepleted T-cell “add-back” to exert an anti-viral infection effect, immunity to EBV and CMV infections was tested at 1 month after infusion in two infected patients, included the one presenting with cerebral and pulmonary lesions. In these two cases, circulating T cells were highly responsive to CMV and EBV as shown by their capacity to secrete -interferon in the presence of an autologous EBV cell line (transfected or not with CMV pp65 antigen) and their capacity to kill CMV pp65-expressing target cells without in vitro restimulation. As shown by CT scan, pulmonary and central nervous system lesions in patient 5 caused by the EBV infection disappeared 11 weeks after the T-cell infusion. For the other patient, CMV antigen was negative 15 weeks post-HSCT (10 weeks postinfusion). Infused allodepleted T cells seem to not recirculate in noninfected patients, an observation that may depend upon the low numbers of infused cells. As illustrated by the three cases of relapse among the five leukemic patients included in the clinical trial, cellular therapy for viral infections is achieved with doses of lymphocytes within the range of the

Exhaustive mutation analysis of the NF1 gene allows identification of 95% of mutations and reveals a high frequency of unusual splicing defects

Neurofibromatosis type 1 (NF1) is one of the most common autosomal dominant disorders and is caused by mutations in the NF1 gene. Mutation detection is complex due to the large size of the NF1 gene, the presence of pseudogenes and the great variety of possible lesions. Although there is no evidence for locus heterogeneity in NF1, mutation detection rates rarely exceed 50%. We studied 67 unrelated NF1 patients fulfilling the NIH diagnostic criteria, 29 familial and 38 sporadic cases, using a cascade of complementary techniques. We performed a protein truncation test starting from puromycin-treated EBV cell lines and, if no mutation was found, continued with heteroduplex, FISH, Southern blot and cytogenetic analysis. We identified the germline mutation in 64 of 67 patients and 32 of the mutations are novel. This is the highest mutation detection rate reported in a study of typical NF1 patients. All mutations were studied at the genomic and RNA level. The mutational spectrum consisted of 25 nonsense, 12 frameshift, 19 splice mutations, six missense and/or small in-frame deletions, one deletion of the entire NF1 gene, and a translocation t(14;17)(q32;q11.2). Our data suggest that exons 10a-10c and 37 are mutation-rich regions and that together with some recurrent mutations they may account for almost 30% of the mutations in classical NF1 patients. We found a high frequency of unusual splice mutations outside of the AG/GT 5¢ and 3¢ splice sites. As some of these mutations form stable transcripts, it remains possible that a truncated neurofibromin is formed. Hum Mutat 15:541–555, 2000. © 2000 Wiley-Liss, Inc.

Exhaustive mutation analysis of the NF1 gene allows identification of 95% of mutations and reveals a high frequency of unusual splicing defects.

Neurofibromatosis type 1 (NF1) is one of the most common autosomal dominant disorders and is caused by mutations in the NF1 gene. Mutation detection is complex due to the large size of the NF1 gene, the presence of pseudogenes and the great variety of possible lesions. Although there is no evidence for locus heterogeneity in NF1, mutation detection rates rarely exceed 50%. We studied 67 unrelated NF1 patients fulfilling the NIH diagnostic criteria, 29 familial and 38 sporadic cases, using a cascade of complementary techniques. We performed a protein truncation test starting from puromycin-treated EBV cell lines and, if no mutation was found, continued with heteroduplex, FISH, Southern blot and cytogenetic analysis. We identified the germline mutation in 64 of 67 patients and 32 of the mutations are novel. This is the highest mutation detection rate reported in a study of typical NF1 patients. All mutations were studied at the genomic and RNA level. The mutational spectrum consisted of 25 nonsense, 12 frameshift, 19 splice mutations, six missense and/or small in-frame deletions, one deletion of the entire NF1 gene, and a translocation t(14;17)(q32;q11.2). Our data suggest that exons 10a-10c and 37 are mutation-rich regions and that together with some recurrent mutations they may account for almost 30% of the mutations in classical NF1 patients. We found a high frequency of unusual splice mutations outside of the AG/GT 5 cent and 3 cent splice sites. As some of these mutations form stable transcripts, it remains possible that a truncated neurofibromin is formed.

WASP Levels in Platelets and Lymphocytes of Wiskott-Aldrich Syndrome Patients Correlate with Cell Dysfunction

Wiskott-Aldrich syndrome, an inherited blood cell disorder due to mutations of the X-chromosome gene WASP (Wiskott-Aldrich syndrome protein), was characterized originally by thrombocytopenia, immunodeficiency, and eczema. Whereas platelet dysfunction is severe and consistent, immune defects are clinically variable, ranging from negligible to life threatening. To understand this heterogeneity, we quantified WASP in PBMC and platelets, and also in neutrophils, of patients with diverse mutations. A surprisingly complex pattern of WASP expression found for lymphoid cells formed the basis for dividing the patient mutations into four groups. Group A have low WASP levels in PBMC and higher levels in EBV cell lines, as well as near normal WASP RNA levels (7 patients, most with mild disease), suggesting that group A WASP molecules are hypersusceptible to proteolysis. Group B have low WASP levels in PBMC and EBV cells and similar low RNA levels (2 patients, moderate disease). Group C have discordant expression: WASP-positive peripheral T cells and WASP-negative peripheral B cells and EBV cell lines (9 patients, variable disease severity). Noteworthy among group C kindred are several instances of B cell lymphomas. In group D, PBMC and EBV cell lines are WASP negative (7 patients, severe disease). In contrast to the complex lymphoid cell expression patterns, all patient platelets examined were WASP negative (18 diverse patients). WASP absence in platelets provides an apparent molecular explanation for the universally severe platelet dysfunction in this disease, and the cumulative lymphoid cell findings suggest that WASP levels play a substantial role in determining immune outcome.

Isolation of broadly reactive, tumor-specific, HLA class-I restricted CTL from blood lymphocytes of a breast cancer patient

Blood lymphocytes of a HLA-A2 positive breast cancer patient were stimulated with either MCF-7 or MDA-MB-231, i.e., HLA-A2-matched allogeneic breast carcinoma cell lines. Several CD8 + CTL clones with reactivity against the stimulator cells but not against K562 were generated. Reactivity could be blocked with monoclonal antibody (mAb) W6/32, MA2.1, and/or BB7.2, indicating that the clones are HLA-class I and HLA-A2 restricted. The CTL clones generated following stimulation with MCF-7, recognized various other allogeneic HLA-A2 + tumor cell lines, including breast carcinoma, renal cell carcinoma, and melanoma cell lines, but not HLA-A2 − tumor cell lines. The CTL clones did not recognize normal HLA-A2 + cells including breast epithelial cells, renal proximal tubular epithelial cells (PTEC), or EBV-transformed B cells including the autologous EBV cell line. In contrast to the CTL clones induced with MCF-7, the reactivity of the clones stimulated with MDA-MB-231, was limited to the stimulator cell MDA-MB-231. Cytotoxicity assays utilizing T2 cells loaded with peptides as target cells indicated that none of the examined CTL-epitopes derived from HER-2/neu, Muc-1, Ep-CAM-1, and p53 were recognized by the CTL clones generated. Our findings underscore that breast cancer is an immunogenic tumor and that HLA-class I-matched allogeneic tumor cells can be used as stimulator cells to generate tumor-specific CTL from peripheral blood of a breast cancer patient with specificity for an antigenic determinant that is broadly expressed on tumor cells from various origins or with specificity limited to the breast cancer stimulator cell.

The Epstein-Barr virus major latent promoter Qp is constitutively active, hypomethylated, and methylation sensitive.

Epstein-Barr virus (EBV) nuclear antigen 1 (EBNA1) is indispensable for viral DNA replication and episome maintenance in latency. Four promoters, Cp, Wp, Qp, and Fp, are known to drive EBNA1 expression. Here we show that the TATA-less Qp is constitutively active in a variety of EBV-positive [EBV(+)] tumors and cell lines, irrespective of the activities of other EBNA1 promoters, the type of viral latency, and the cell type. The transcription of highly regulated promoters such as the EBV Cp is known to be directly regulated by CpG methylation. To characterize the role of CpG methylation in the regulation of the constitutively active Qp, we performed bisulfite genomic sequencing and functional analyses using a methylation cassette transcriptional reporter assay. Twenty consecutive CpG sites (16 proximal to the Qp initiation site and 4 upstream of the adjacent Fp initiation site) were studied by bisulfite sequencing of DNA extracted from EBV(+) tumors and cell lines. Eighteen EBV(+) tumors of lymphoid (B, T, and NK cell) or epithelial origin and five Burkitt's lymphoma cell lines were studied. The 16 CpG sites proximal to Qp were virtually all unmethylated, but the 4 CpG sites upstream of the Fp initiation site were variably methylated. The methylation cassette assay showed that in vitro methylation of the Qp cassette (-172 to +32) resulted in strong repression of Qp activity in transient transfection. Thus, Qp is susceptible to repression by methylation but was found to be consistently hypomethylated and expressed in all tumors and tumor-derived cell lines studied.