EBV-transformed Cell Lines Research Articles

Effects of Epstein-Barr Virus Latent Membrane Protein 2A (LMP2A) on cytokine production in primary and transformed B cells (105.40)

Abstract Epstein-Barr Virus (EBV) latently-infected B cells are precursors of EBV-associated malignancies. However, the transition from a latently-infected cell to a malignant tumor cell is poorly understood. Studies show that EBV modulates pro-survival and anti-inflammatory cytokine levels in EBV-transformed cell lines. However, gene expression in these cell lines may not reflect gene expression found in latent B cells and lymphomas. One EBV protein, LMP2A, is consistently detected in both latently-infected resting B cells, as well as in EBV-associated malignancies. Therefore, we hypothesized that LMP2A may modulate cytokines that aid in the transition between latency and malignancy. Cytokine array data analyzing B cells from LMP2A-transgenic mice and B cell lymphomas expressing LMP2A indicate that LMP2A does not globally alter B cell-produced cytokine levels, but rather targets a limited number of cytokines. Additional studies using ELISA and Real-time RT-PCR confirm that LMP2A increases IL-10 levels and flow cytometry reveals that LMP2A increases the amount of IL-10 produced per B cell. Inhibitors of the LMP2A signaling pathway are being tested to determine the signaling requirements for LMP2A-mediated increases in IL-10. These data identify a novel function of LMP2A in the alteration of a cytokine that is important for both tumor survival and anti-tumor responses.

Abstract 693: Development of a protein-based functional assay for the determination of BRCA1 carrier status

Abstract While most breast cancers are sporadic, 5-10% are hereditary and attributable to the presence of a mutation in the breast cancer associated gene 1 (BRCA1). The BRCA1 gene encodes a tumour suppressor with several well described roles in maintaining genomic integrity, such as cell cycle checkpoint control and DNA repair. BRCA1 is also known to play a role in the differentiation of breast epithelial cells. Given the high risk of cancer development associated with BRCA1 mutation carriers, it is important that they be identified early and accurately. Previous work in our lab developed a novel functional assay to predict BRCA1 status based on gene expression profiles. That study analyzed EBV-transformed lymphocyte cell lines (LCLs) of BRCA1 mutation carriers (BRCA1+/−) and controls (BRCA1+/+) and found that lymphocytes from BRCA1 carriers could be distinguished with high fidelity (∼90% accuracy) from individuals with two wild type copies of the gene. Interestingly, it was found that many of the genes used to distinguish BRCA1 mutation carriers were also markers of differentiation in blood cells. We are now using this information to develop a flow cytometric protein-based biomarker assay. To do this, our transcriptome microarray data was analyzed to identify genes with strong edictability of BRCA1 status, including genes with known roles in lymphocyte differentiation such as TBX21 and CXCR3. These genes were then used to model the assay in-silico using a training set of data comprised of 53 LCLs. The model was designed by selecting genes both up- and down-regulated in BRCA1 carriers, placing them in pairs and looking at the ratio of their expression values. This model was subsequently validated using a test data set comprising 16 LCLs. Overall accuracy of the model was 94%, correctly predicting 54/59 LCLs. Based on in-silico modelling, we chose six antibodies to develop the assay. To identify other candidate proteins, LCL lysates were analyzed by mass spectrometry (MS). The results identified several other candidate proteins for assay development including IgG1, IgD, BCR and MX1. Interestingly, as was the case in our transcriptome analysis, these MS identified proteins also suggested that BRCA1 mutation carriers displayed defects in cellular differentiation. This was indicated by the higher expression of surface markers in responding to less differentiated B-lymphocytes in comparison with controls. Ultimately, the assay will be applied to fresh patient blood samples with the expectation that it will become an assay that can be implemented in a clinical setting to provide accurate identification of BRCA1 mutation carriers and aid in disease management. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 693. doi:1538-7445.AM2012-693

Methylome-wide comparison of human genomic DNA extracted from whole blood and from EBV-transformed lymphocyte cell lines

DNA from Epstein-Barr virus-transformed lymphocyte cell lines (LCLs) has proven useful for studies of genetic sequence polymorphisms. Whether LCL DNA is suitable for methylation studies is less clear. We conduct a genome-wide methylation investigation using an array set with 45 million probes to investigate the methylome of LCL DNA and technical duplicates of WB DNA from the same 10 individuals. We focus specifically on methylation sites that show variation between individuals and, therefore, are potentially useful as biomarkers. The sample correlations for the methylation variable probes ranged from 0.69 to 0.78 for the WB duplicates and from 0.27 to 0.72 for WB vs LCL. To compare the pattern of the methylation signals, we grouped adjacent probes based on their inter-correlations. These analyses showed ∼29 000 and ∼14 000 blocks in WB and LCL, respectively. Merely 31% of the methylated regions detected in WB were detectable in LCLs. Furthermore, we observed significant differences in mean difference between WB and LCL as compared with duplicates of WB (P-value =2.2 × 10(-16)). Our study shows that there are substantial differences in the DNA methylation patterns between LCL and WB. Thus, LCL DNA should not be used as a proxy for WB DNA in methylome-wide studies.

Heat shock factor 1 upregulates transcription of Epstein–Barr Virus nuclear antigen 1 by binding to a heat shock element within the BamHI-Q promoter

Epstein–Barr virus (EBV) nuclear antigen 1 (EBNA1) is essential for maintenance of the episome and establishment of latency. In this study, we observed that heat treatment effectively induced EBNA1 transcription in EBV-transformed B95-8 and human LCL cell lines. Although Cp is considered as the sole promoter used for the expression of EBNA1 transcripts in the lymphoblastoid cell lines, the RT-PCR results showed that the EBNA1 transcripts induced by heat treatment arise from Qp-initiated transcripts. Using bioinformatics, a high affinity and functional heat shock factor 1 (HSF1)-binding element within the − 17/+4 oligonucleotide of the Qp was found, and was determined by electrophoretic mobility shift assay and chromatin immunoprecipitation assay. Moreover, heat shock and exogenous HSF1 expression induced Qp activity in reporter assays. Further, RNA interference-mediated HSF1 gene silencing attenuated heat-induced EBNA1 expression in B95-8 cells. These results provide evidence that EBNA1 is a new target for the transcription factor HSF1.

Anti-Viral Cytotoxic T-Lymphocyte Therapy

AbstractAbstract SCI-50Cytomegalovirus (CMV), Epstein Barr virus (EBV) and adenovirus (Ad) are particularly problematic in patients after hematopoietic stem cell transplantation (HSCT) and are associated with significant morbidity and mortality. While antiviral pharmacotherapy may help prevent or treat CMV or Ad, and CD20-specific antibodies may control EBV-associated lymphoproliferation, these drugs are expensive, toxic and often ineffective due to primary or secondary resistance. These deficiencies in conventional therapeutics have increased interest in an immunotherapeutic approach to viral disorders. Adoptive transfer of T-cells in the form of donor lymphocyte infusions (DLI) has been used to treat viral infection after allogeneic HSCT, but has so far proved to be of limited effectiveness for patients with viral infections such as Ad, and often produce GvHD. Adoptive transfer of peripheral blood derived virus-specific cytotoxic T lymphocytes (CTL) directed to CMV, EBV and Ad can rapidly reconstitute anti-viral immunity post-HSCT without causing GvHD. We have shown that peripheral blood-derived T-cell lines enriched in cells recognizing CMV, EBV and Ad can reproducibly control infections due to all three viruses after allogeneic HSCT. This study demonstrated that the multivirus-specific T cells expand in vivo and are active against all three viruses. Furthermore, by restoring immunity to three viruses simultaneously, the need for continued prophylaxis with pharmacotherapy is eliminated, thus, improving the efficiency and cost effectiveness of protecting HSCT recipients from these potentially lethal viruses. In principle, this strategy could be applied with comparable success to recipients of cord blood (CB) transplants; however, certain obstacles to the extension of this approach must be circumvented. These include: (i) the limited numbers of CB T-cells available for manipulation and (ii) the naivety of CB T-cells. Hence, the development of virus-protective T-cell therapy for patients undergoing CBT requires the priming and extensive expansion of naïve T-cells rather than the more limited and simple direct expansion of pre-existing virus-specific memory T-cell populations from virus-experienced donors. Further, CB T-cells have lower cytotoxic activity and higher activation-induced cell death than peripheral blood T-cells. These limitations have prevented the production of virus-specific cord blood-derived CTL in sufficient numbers for clinical use. Recent studies demonstrate that ex vivo priming of naïve T-cells can be achieved in the presence of combinations of soluble cytokines such as IL-7, IL-15, and IL-12 that respectively decrease the antigen concentration threshold, direct T-cells towards a central memory phenotype, and influence the polarization of Th1 cells. We have now shown that Ad5f35pp65-transduced CB-derived APC can be used to generate large numbers of autologous T-cells specific for CMV and Ad, even from the phenotypically naive T-cell subpopulation. Addition of EBV-transformed B-lymphoblastoid cell lines (LCL) to the APCs allows the Ad/CMV specificity of the CB T-cells to be extended to EBV. In addition, the multivirus-specific T-cells recognize an array of epitopes after only 2 weeks expansion in vivo. We have now initiated a clinical trial using CB-derived multivirus-specific T cells to infuse to patients after CB transplant. Moreover, we propose that these virus-specific T cells generated from cord blood or peripheral blood can be genetically modified to (i) redirect specificity to leukemia as well as viral antigens and (ii) overcome tumor immune evasion strategies. Disclosures:No relevant conflicts of interest to declare.

Synergistic effects of CTLA‐4 blockade with tremelimumab and elimination of regulatory T lymphocytes in vitro and in vivo

Anti-CTLA-4 monoclonal antibodies (mAb) that block the interaction of CTLA-4 with CD80 and CD86 such as tremelimumab and ipilimumab are currently being tested in the clinic for cancer treatment exploiting their properties to de-repress tumor-specific cellular immunity. Addition of the fully human anti-CTLA-4 (tremelimumab) to cultures of human T cells with allogenic dendritic cells (DCs) did not increase proliferation. Magnetic bead-mediated elimination of CD4(+) CD25(+) regulatory T cells (T(reg)) before setting up those alloreactive cultures also largely failed to increase primary proliferation. In contrast, predepletion of CD4(+) CD25(+) T(reg) and culture in the presence of tremelimumab synergistically resulted in increased proliferation and DC:T-cell aggregation. These effects were much more prominent in CD4 than in CD8 T cells. The synergy mechanism can be traced to enhanced CTLA-4 expression in effector cells as a result of T(reg) elimination, thereby offering more targets to the blocking antibody. Human T cells and allogenic DCs (derived both from healthy donors and advanced cancer patients) were coinjected in the peritoneum of Rag2(-/-) IL-2Rγ(-/-) mice. In these conditions, tremelimumab injected intravenously did not significantly enhance alloreactive proliferation unless T(reg) cells had been predepleted. Synergistic effects in vivo were again largely restricted to the CD4 T-cell compartment. In addition, T(reg) depletion and CTLA-4 blockade synergistically enhanced specific cytotoxicity raised in culture against autologous EBV-transformed cell lines. Taken together, these experiments indicate that tremelimumab therapy may benefit from previous or concomitant T(reg) depletion.

PTEN transcript variants caused by illegitimate splicing in “aged” blood samples and EBV-transformed cell lines

PTEN is one of the most frequently mutated tumor suppressor genes in human cancers. Mutations occur in either heritable or sporadic fashion. Sequencing of cDNA from patients and normal individuals often reveals splicing variants (SVs) of PTEN, some of which are non-mutation related. To investigate whether these SVs were the result of illegitimate splicing (a general decrease of fidelity in splicing site selection in “aged” samples), we tested “aged” blood from individuals who had normal PTEN transcripts in their “fresh” mononuclear cells. Blood from 20 normal individuals was collected and split into two aliquots. Total RNA and DNA were extracted immediately (“fresh”) and 48 h later (“aged”), respectively. Using RT-PCR, subcloning and sequencing, we found seven types of SVs. No mutation was detected in the related intron–exon flanking region in genomic DNA in either “fresh” or “aged” samples. Some of the SVs were also consistently present in both the “fresh” and “aged” EBV-transformed lymphoblastoid cells from six normal individuals. Western blot data indicated that the PTEN protein level (in full length) was not altered in the “fresh” EBV-transformed lymphoblastoid cells with SVs. In conclusion, our data demonstrate that PTEN illegitimate splicing often occurs in “aged” blood and EBV-transformed lymphoblastoid cells. Therefore, it is critical to note the time point of RNA extraction when investigating for PTEN aberrant transcripts. We hope that our data will increase awareness about the sample status, because gene expression data may be potentially flawed from “aged” samples, particularly when dealing with clinical samples.

Abstract SSY01-02: Notch and radiation resistance in glioma stem cells

Abstract SSY01-02: Notch and radiation resistance in glioma stem cells

Abstract SSY01-04: Discovery and validation of genome-wide genetic signatures of chemotherapy susceptibility: A translational model

Abstract SSY01-04: Discovery and validation of genome-wide genetic signatures of chemotherapy susceptibility: A translational model

Overview of the Type I Diabetes Genetics Consortium

The Type I Diabetes Genetics Consortium (T1DGC) is an international, multicenter research program with two primary goals. The first goal is to identify genomic regions and candidate genes whose variants modify an individual's risk of type I diabetes (T1D) and help explain the clustering of the disease in families. The second goal is to make research data available to the research community and to establish resources that can be used by, and that are fully accessible to, the research community. To facilitate the access to these resources, the T1DGC has developed a Consortium Agreement (http://www.t1dgc.org) that specifies the rights and responsibilities of investigators who participate in Consortium activities. The T1DGC has assembled a resource of affected sib-pair families, parent-child trios, and case-control collections with banks of DNA, serum, plasma, and EBV-transformed cell lines. In addition, both candidate gene and genome-wide (linkage and association) studies have been performed and displayed in T1DBase (http://www.t1dbase.org) for all researchers to use in their own investigations. In this supplement, a subset of the T1DGC collection has been used to investigate earlier published candidate genes for T1D, to confirm the results from a genome-wide association scan for T1D, and to determine associations with candidate genes for other autoimmune diseases or with type II diabetes that may be involved with beta-cell function.

5'Nucleotidase (NT5C2) Genotype Influences Leukemic Blast Concentration of Ara-CTP in Pediatric Patients with Acute Myeloid Leukemia.

Abstract 593▪▪This icon denotes an abstract that is clinically relevant.Cytarabine (ara-C) is one of the most widely used chemotherapeutic agents in the treatment of acute myeloid leukemia (AML). Inter-patient variability in clinical response following ara-C treatment has been correlated with leukemic cell concentration of ara-C triphosphate (ara-CTP), the active metabolite of the drug. NT5C2 is an inactivating enzyme in the activation pathway of ara-C, and catalyzes conversion of ara-C monophosphate to ara-C. We identified genetic variations in NT5C2 and investigated the association of NT5C2 genetic variations with its mRNA expression and with intracellular ara-CTP accumulation. The coding exons of NT5C2 were resequenced using genomic DNA from EBV-transformed B-lymphoblastoid HapMap cell lines derived from 90 CEPH (representing 30 trios with European ancestry) and 90 YRI (30 trios with African ancestry) sample. Thirty-nine genetic variants (one insertion-deletion and 38 SNPs), including three nonsynonymous SNPs (Thr3Ala, Lys47Arg, Gln136Arg), were identified. The strongly linked SNPs (R2 > 0.8) in CEPH and YRI were grouped into ten distinct groups. NT5C2 mRNA expression levels were determined in all samples and was significantly associated with ethnicity (p = 8.5 × 10-6), with subjects of African ancestry having significantly higher NT5C2 mRNA expression as compared to the subjects with European ancestry (Figure A). Several NT5C2 SNPs were associated with its mRNA expression in both the ethnic groups. [Display omitted] To determine the clinical implication of NT5C2 SNPs, we investigated selected germline NT5C2 SNPs in pediatric AML patients treated on the St Jude AML 97 protocol (n=55). Patients were randomly assigned to receive ara-C as either a short daily infusion (500 mg/m2/dose intravenously over 2 hrs daily for 5 days) or a continuous infusion (500 mg/m2/day as a continuous infusion over 5 days). Bone marrow was collected at the end of the ara-C infusion on day1 for patients receiving the short daily infusion (n=27), and at 10 hrs after the start of the infusion for those receiving the continuous infusion (n=28). Ara-CTP levels in leukemia cells were analyzed by HPLC. Intracellular accumulation of ara-CTP was significantly higher when given as short daily infusion, as compared to continuous infusion (0.56 ±0.5 vs. 0.33± 0.31 nmol ara-CTP/2×107leukemic cells; p = 0.014). The inter-patient variability for blast ara-CTP concentration was 40-fold in the short infusion arm and 101-fold in continuous infusion arm. Karyotypes, gender, age, and ethnicity had no significant influence on the leukemic ara-CTP levels. NT5C2 Group 1 SNPs were found to be significantly associated with intracellular ara-CTP levels in leukemic blasts from ara-C-treated Pediatric AML Patients (Figure B). These results suggest that genetic variation in NT5C2 could influence its activity and/or expression and may predict the variability observed in intracellular levels of the ara-C active metabolite, ara-CTP, which in turn can influence treatment response. Disclosures:No relevant conflicts of interest to declare.

High-throughput isolation of immunoglobulin genes from single human B cells and expression as monoclonal antibodies

Defining human B cell repertoires to viral pathogens is critical for design of vaccines that induce broadly protective antibodies to infections such as HIV-1 and influenza. Single B cell sorting and cloning of immunoglobulin (Ig) heavy- and light-chain variable regions (V H and V L) is a powerful technology for defining anti-viral B cell repertoires. However, the Ig-cloning step is time-consuming and prevents high-throughput analysis of the B cell repertoire. Novel linear Ig heavy- and light-chain gene expression cassettes were designed to express Ig V H and V L genes isolated from sorted single B cells as IgG1 antibody without a cloning step. The cassettes contain all essential elements for transcriptional and translational regulation, including CMV promoter, Ig leader sequences, constant region of IgG1 heavy- or Ig light-chain, poly(A) tail and substitutable V H or V L genes. The utility of these Ig gene expression cassettes was established using synthetic V H or V L genes from an anti-HIV-1 gp41 mAb 2F5 as a model system, and validated further using V H and V L genes isolated from cloned EBV-transformed antibody-producing cell lines. Finally, this strategy was successfully used for rapid production of recombinant influenza mAbs from sorted single human plasmablasts after influenza vaccination. These Ig gene expression cassettes constitute a highly efficient strategy for rapid expression of Ig genes for high-throughput screening and analysis without cloning.

An Sp1/Sp3 Binding Polymorphism Confers Methylation Protection

Hundreds of genes show aberrant DNA hypermethylation in cancer, yet little is known about the causes of this hypermethylation. We identified RIL as a frequent methylation target in cancer. In search for factors that influence RIL hypermethylation, we found a 12-bp polymorphic sequence around its transcription start site that creates a long allele. Pyrosequencing of homozygous tumors revealed a 2.1-fold higher methylation for the short alleles (P<0.001). Bisulfite sequencing of cancers heterozygous for RIL showed that the short alleles are 3.1-fold more methylated than the long (P<0.001). The comparison of expression levels between unmethylated long and short EBV-transformed cell lines showed no difference in expression in vivo. Electrophorectic mobility shift assay showed that the inserted region of the long allele binds Sp1 and Sp3 transcription factors, a binding that is absent in the short allele. Transient transfection of RIL allele-specific transgenes showed no effects of the additional Sp1 site on transcription early on. However, stable transfection of methylation-seeded constructs showed gradually decreasing transcription levels from the short allele with eventual spreading of de novo methylation. In contrast, the long allele showed stable levels of expression over time as measured by luciferase and ∼2–3-fold lower levels of methylation by bisulfite sequencing (P<0.001), suggesting that the polymorphic Sp1 site protects against time-dependent silencing. Our finding demonstrates that, in some genes, hypermethylation in cancer is dictated by protein-DNA interactions at the promoters and provides a novel mechanism by which genetic polymorphisms can influence an epigenetic state.

Antitumor Activity of Immunotoxins with T-Cell Receptor–like Specificity against Human Melanoma Xenografts

In this study, we have explored the use of Fab-toxin proteins (immunotoxin) to target antigen-specific MHC-peptide complexes of in vitro and in vivo cancer cells. A human phage display library was used to screen for T-cell receptor (TCR)-like antibodies that are highly specific for the peptide melanoma-associated antigen MART-1(26-35) presented by HLA-A201. We also used previously selected TCR-like antibodies specific for the peptide melanoma-associated antigen gp100(280-288) presented by HLA-A201. The recombinant immunotoxin constructs were generated by fusing the targeting Fab fragment to a truncated form of Pseudomonas exotoxin, PE38KDEL. These immunotoxins bound with high affinity to the EBV-transformed JY cell line pulsed with the aforementioned peptides and internalized within 30 min. A significant inhibition of protein synthesis, which resulted in cell death, was detected at 24 h. MART-1-specific and gp100-specific immunotoxins bound and killed HLA-A201 melanoma MART-1(+) and gp100(+) cell lines that were presented at natural levels but do not bind to HLA-A201(-) or to HLA-A201(+) MART-1(-) and gp100(-) cell lines. In severe combined immunodeficient mice, MART-1 and gp100 immunotoxins significantly and discriminately inhibited human melanoma growth. These results show that MHC class I/peptide complexes can serve as a specific target for passive immunotherapy of cancer.

Characterization and large production of human monoclonal antibodies against the HIV-1 envelope

Peripheral blood lymphocytes from a volunteer immunized with a recombinant vaccinia virus VSC-25 expressing the gp160 env protein of HTLV-IIIB strain and from an asymptomatic HIV-infected individual were immortalized by Epstein-Barr (EBV). Clones which secrete human monoclonal antibodies from the two individuals (DZ, IgG1, lambda and C31, IgG1, kappa) were obtained and were stable for more than 2 years. The two monoclonals were directed against the gp160 env protein of HIV, DZ directed against the gp41 and C31 directed against the gp120. C31 was group-specific, whereas DZ was directed against the HTLV-IIIB and HTLV-RF strains. The epitope recognized by DZ was mapped to the carboxy terminus of the gp41, by expression of HIV DNA fragments in a yeast system and peptide analysis. The C31 epitope was not expressed by the yeast library and not present among the peptides which were tested. Monoclonal antibodies had no inhibitory effect in an HIV-induced cell fusion assay, but DZ showed a weak neutralizing activity against the HTLV-IIIB strain. Cloned EBV-transformed cell lines were fused to a murine myeloma, which allowed the heteromyeloma to be cultivated in serum-free medium. The monoclonal antibodies were produced in large quantity in a hollow-fibre reactor at defined culture conditions and purification procedures.

14C1, an antigen associated with human ovarian cancer, defined using a human IgG monoclonal antibody

We have selected a human EBV-transformed cell line from the involved lymph nodes of an ovarian cancer patient which secretes an IgG1 kappa antibody, able to recognize an antigen present on the surface of ovarian cancer cells. The antigen, termed '14Cl,' has previously been shown by immunohistological techniques to be present on the surface of the malignant cells within tumour specimens. Western blotting analysis has shown that the majority of primary ovarian cancer specimens and three continuous cell lines derived therefrom express 14Cl; other tissue types were negative. Preliminary biochemical characterization has been carried out, which shows that the 14Cl antigen has a molecular weight range of 25-32 kD and an isoelectric point from pI 6.3 to 6.8. We believe that the 14Cl antigen is immunologically relevant to ovarian cancer patients and may therefore represent a novel target for both active and passive immunotherapy.

C1GALT1C1 (COSMC) Mutations in Four Individuals with Tn+ Phenotype.

Tn is a cryptantigen located on O-linked oligosaccharides of cell membrane glycoproteins and is composed of N-acetylgalactosamine a -linked to serine or threonine of the protein backbone. Tn is expressed on hemopoietic cells of individuals with the rare idiopathic Tn syndrome, characterized by a variable pattern of Tn expression suggestive of its somatic and clonal origin. Tn is also associated with overexpression in several autoimmune diseases and malignancies. Tn is a bioprecursor of the T cryptantigen and subsequently the disialotetrasaccharide units typical of O-linked oligosaccharides of RBC sialoglycoproteins. Tn results from defective oligosaccharide biosynthesis caused by the malfunction of T-synthase, a b1,3-galactosyltransferase. Recent evidence suggested that the activity of T-synthase is dependent on a molecular chaperone, Cosmc (Ju and Cummings, PNAS 2002; 99:16613–18). In view of this evidence, we investigated whether Cosmc is required for T-synthase activity and ultimately for Tn phenotype by obtaining material from 4 apparently healthy, unrelated Caucasian individuals with Tn phenotype. Case 1 was 93.9% Tn+ on an EBV-transformed lymphoblastoma cell line. Analysis of the Cosmc gene, C1GALT1C1, showed a homozygous 428C>T, Ala143Val mutation and Case 1 showed a complete lack of expression of C1GALT1C1 cDNA. Case 2 revealed no C1GALT1C1 mutations in DNA extracted from plasma, but in DNA of lymphocyte origin an apparent heterozygous 454G>A, Glu152Lys change was observed, in agreement with Ju and Cummings (Nature 2005, 437:1252). When Case 2 hemopoietic progenitor cells were expanded into the erythroid cell line, the mutation appeared homozygous. Tn expression varied from 76.1% in lymphocytes, 90.7% in RBCs to 96.9% in erythroblasts. Case 3 showed 19.4% Tn+ lymphocytes, compared to 97.0% Tn+ RBCs. In Case 3 we found a single point mutation 577T>C, Ser193Pro. Case 4 exhibited 46.5% Tn+ lymphocytes, 96.0% Tn+ RBCs and 90.2% Tn+ erythroblasts. DNA analysis revealed 3G>C, converting the translation-initiating methionine to isoleucine and predicting the loss of first 12 amino acids of the protein, potentially altering its morphology. The mutations found in Cases 2–4 reflected the clonal nature of Tn, appearing heterozygous in DNA of lymphocyte origin and homozygous in DNA of erythroid origin. To confirm that the observed mutations are indeed responsible for Tn phenotype, pBabe puro vector with Tn or wild type C1GALT1C1 inserts was transfected into Jurkat cells. Untransfected cells and cells transfected with bare vector expressed Tn. Jurkat cells transfected with wild-type C1GALT1C1 were Tn-negative while those transfected with C1GALT1C1 from Cases 2–4 expressed Tn. From this evidence we postulate that Cosmc is directly involved in the expression of Tn phenotype. To investigate the involvement of other genes, we performed expression profiling of 3 Tn and 4 control samples hybridized to HG-U133A arrays. A list of 100 up-regulated and 173 down-regulated genes, with 1.5× fold difference in expression, was obtained. Some genes, relating to erythrocyte development/heme biosynthesis were upregulated, while down-regulated genes were related to cholesterol/lipid metabolism. Real-time Q-PCR on six differentially expressed genes of interest, down-regulated FABP5, CYP1B1 and LRP8 and up-regulated AQP1, AQP3 and EPB42, confirmed the microarray results, and elevated expression of AQP3 on Tn + RBCs was detected serologically. The effects of C1GALT1C1 mutations are wider than Tn expression on hemopoietic cells.

A pedigree with autosomal dominant thrombocytopenia, red cell macrocytosis, and an occurrence of t(12:21) positive pre-B acute lymphoblastic leukemia

Sampling and analyzing new families with inherited blood disorders are major steps contributing to the identification of gene(s) responsible for normal and pathologic hematopoiesis. Familial occurrences of hematological disorders alone, or as part of a syndromic disease, have been reported, and for some the underlying genetic mutation has been identified. Here we describe a new autosomal dominant inherited phenotype of thrombocytopenia and red cell macrocytosis in a four-generation pedigree. Interestingly, in the youngest generation, a 2-year-old boy presenting with these familial features has developed acute lymphoblastic leukemia characterized by a t(12;21) translocation. Tri-lineage involvement of platelets, red cells and white cells may suggest a genetic defect in an early multiliear progenitor or a stem cell. Functional assays in EBV-transformed cell lines revealed a defect in cell proliferation and tubulin dynamics. Two candidate genes, RUNX1 and FOG1, were sequenced but no pathogenic mutation was found. Identification of the underlying genetic defect(s) in this family may help in understanding the complex process of hematopoiesis.

Effect of population and gender on chemotherapeutic agent–induced cytotoxicity

Large interindividual variance is observed in both response and toxicity associated with chemotherapy. Our goal is to identify factors that contribute to chemotherapy-induced toxicity. To this end, we used EBV-transformed B-lymphoblastoid HapMap cell lines derived from 30 Yoruban trios (African descent) and 30 Centre d' Etude du Polymorphisme Humain (CEPH) trios (European descent) to evaluate population- and gender-specific differences in cytotoxicity of carboplatin, cisplatin, daunorubicin, and etoposide using a high-throughput, short-term cytotoxicity assay. The IC(50) was compared for population- and gender-specific differences for the four drugs. We observed large interindividual variance in IC(50) values for carboplatin, cisplatin, daunorubicin, and etoposide for both Yoruban and CEPH populations (range from 8- to 433-fold). Statistically significant differences in carboplatin and daunorubicin IC(50) were shown when comparing Yoruban cell lines (n = 89) to CEPH cell lines (n = 87; P = 0.002 and P = 0.029, respectively). This population difference in treatment induced cytotoxicity was not seen for either cisplatin or etoposide. In the Yoruban population, cell lines derived from females were less sensitive to platinating agents than males [median carboplatin IC(50), 29.1 versus 24.6 micromol/L (P = 0.012); median cisplatin IC(50), 7.0 versus 6.0 micromol/L (P = 0.020) in female and male, respectively]. This difference was not observed in the CEPH population. These results show that population and gender may affect risk for toxicities associated with certain chemotherapeutic agents.

Functional Polymorphism of the KIR3DL1/S1 Receptor on Human NK Cells

NK cells express both inhibitory and activatory receptors that allow them to recognize target cells through HLA class I Ag expression. KIR3DL1 is a receptor that recognizes the HLA-Bw4 public epitope of HLA-B alleles. We demonstrate that polymorphism within the KIR3DL1 receptor has functional consequences in terms of NK cell recognition of target. Inhibitory alleles of KIR3DL1 differ in their ability to recognize HLA-Bw4 ligand, and a consistent hierarchy of ligand reactivity can be defined. KIR3DS1, which segregates as an allele of KIR3DL1, has a short cytoplasmic tail characteristic of activatory receptors. Because it is very similar to KIR3DL1 in the extracellular domains, it has been assumed that KIR3DS1 will recognize a HLA-Bw4 ligand. In this study, we demonstrate that KIR3DS1 is expressed as a protein at the cell surface of NK cells, where it is recognized by the Z27 Ab. Using this Ab, we found that KIR3DS1 is expressed on a higher percentage of NK cells in KIR3DS1 homozygous compared with heterozygous donors. In contrast to the inhibitory KIR3DL1 allotypes, KIR3DS1 did not recognize HLA-Bw4 on EBV-transformed cell lines.