B-lymphoblastoid Cell Lines Research Articles

PS1320 CDCA7 REGULATES LYMPHOMA CELLS MIGRATION, INVASIVENESS AND THEIR MICROTUBULE AND ACTOMYOSIN CYTOSKELETONS DYNAMICS

Background:Metastasis is the major cause of death from cancer. Indeed, lymphoid neoplasm metastases are extremely difficult to treat due to their heterogeneity and their frequent affectation of the central nervous system. To acquire metastatic capacity, cells require increasing their migration and invasion capacities. In spite of the relevance of these processes, the mechanisms and genes involved in their regulation are poorly understood.Immortality is essential, but insufficient for tumorigenesis. Certainly, some non‐tumor cells, such as lymphoblastoid B‐cell lines (LCLs), are able to divide indefinitely. To identify genes involved in tumor traits beyond immortalization, our group took the innovative approach of comparing gene expression profiles of B‐cell lymphomas with immortal LCLs. Among >1,600 genes differentially expressed in lymphoma cells, we focused in CDCA7 as one of the most significantly up‐regulated genes.CDCA7 had been identified as a C‐MYC target gene whose mRNA was overexpressed in human tumors, particularly in chronic leukemia. Several studies based in CDCA7 overexpression suggested however that CDCA7 exhibited a limited in vivo transformation capacity. Moreover, its forced expression inhibited C‐MYC‐induced transformation. In contrast, we showed recently that CDCA7 knock‐down in lymphoma and leukemia cells sharply decreased their tumorigenic capacity without affecting their proliferation in liquid culture.Aims:The goal of this work was to investigate the potential role of CDCA7 in lymphoma invasion and migration.Methods:DG‐75, BL‐2, and Toledo lymphoma cell lines were transduced with lentivirus encoding control or CDCA7‐specific shRNA. We used subcutaneous xenografts in NOD‐SCID immunodeficient mice and in the yolk sac of zebrafish embryos to analyze the invasion capacity of lymphoma cells in vivo. We also used transwell permeable plates coated with matrigel or fibronectin to assess the invasion or migration capacity of these cells in vitro. Flow cytometry and quantitative PCR (qPCR) were used to determine the expression of potential mediators of cell migration and invasion, and immunofluorescence confocal imaging was employed to determine the cellular distribution of cytoskeleton components.Results:In the mouse xenograft model, we observed that CDCA7 silencing decreased tumor growth. Subsequent immunohistochemical analysis of the tumors revealed that, contrary to control cells, CDCA7‐silenced cells barely infiltrated and disorganized the neighbor tissues. Moreover, CDCA7 knockdown markedly decreased DG‐75 and Toledo cells invasion in the zebrafish model. Migration and invasion in vitro assays with lentivirally knocked‐down DG‐75, BL‐2, and Toledo lymphoma cells showed that CDCA7 mediates invasion and migration through matrigel‐ and fibronectin‐coated transwells, respectively. As qPCR and flow cytometry analyses revealed that CDCA7 does not regulate the expression of MMP2 and MMP9 or the expression of the integrin α4β1 (the main fibronectin receptor of these cells), we next investigated whether CDCA7 regulated cytoskeleton dynamics. Confocal immunofluorescence imaging analysis revealed that CDCA7 knock‐down markedly increased actin polymerization and myosin light chain phosphorylation, while impairing actin and tubulin cytoskeletons polarization.Summary/Conclusion:Our results strongly suggest that CDCA7 is a critical mediator of lymphoma cells invasion and migration, likely by regulating actomyosin and tubulin cytoskeleton dynamics.

CpG-A and CpG-B oligonucleotides differentially regulate MR1-mediated microbial Ag presentation

Abstract Mucosal-associated invariant T (MAIT) cells are conserved innate T cells that express a semi-invariant T cell receptor (TCR; Vα7.2 in human and Vα19 in mice). MAIT cells are activated by microbial Ags presented by the major histocompatibility complex (MHC)-related molecule, MR1. Little is known how MR1 molecules are processed and loaded with microbial Ag in APCs. We have previously found that Toll-like Receptor 9 (TLR9) is important in regulating MR1-mediated bacterial Ag presentation to MAIT cells in B cells. We further demonstrated that, similar to human B cells, CpG-A (but not CpG-B) upregulated MR1 expression on mouse B cells as well. CpG-A treatment increased MR1 expression on WT mouse B cells but not MR1 KO B cells, further confirming that activation of B cells by CpG-A specifically enhances MR1 surface expression. Initial RNAseq analysis has indicated that B cells activated by CpG-A are distinctively different from those activated by CpG-B or the vehicle control. In conclusion, CpG-A and CpG-B differentially impact MR1 expression and microbial Ag presentation. We have successfully deleted the expression of TLR9 and its downstream mediators in a human B lymphoblastoid cell line (B-LCL) using CRISPR technology. Future work will focus on how genes altered by the activation of CpG-A (but not CpG-B) regulate MR1-mediated microbial Ag presentation.

Lipid composition impacts the assembly of MHC class II molecules in nanodiscs

Abstract Class II major histocompatibility complex molecules (MHCII) are transmembrane glycoproteins that display peptides generated and selected by intracellular antigen processing and presentation mechanisms to CD4+T cells, initiating an adaptive immune response. MHCII is expressed on the cell surface and in endosomal compartments, each of which have their own signature membrane characteristics. The traditional strategy to investigate peptide binding to MHCII has relied on the use of soluble MHCII, in which the transmembrane portion has been removed. This work studied MHCII in its native, membrane-embedded form. To this end, we used nanodisc, a synthetic model membrane device, to evaluate the effect of membrane lipid composition on MHCII assembly. The full-length human MHCII allele HLA-DR1 (DR1) was isolated from B-lymphoblastoid cell lines via immunoaffinity chromatography. Three types of nanodisc were generated: simple, fluid disordered and rigid ordered, each of them characterized by a unique lipid composition. Nanodiscs were separated using fast protein liquid chromatography (FPLC), the readings from which showed clear differences between nanodisc types. We observed that the fluid disordered nanodisc showed DR1 assembly as a cluster, with one major FPLC-generated peak. In contrast, simple and rigid nanodiscs were distinguished by multiple peaks. As DR1 tends to form aggregates, we inferred that in simple and rigid nanodiscs DR1 tetramers and dimers form in separate nanodisc assemblies. Taken together, our results indicate that membrane lipid composition has a strong impact on native MHCII assembly. As such, we predict that MHCII conformation and activity are function of the cell compartment they reside at any point in time.

CD19 chimeric antigen receptor (CAR) engineered epstein-barr virus (EBV) specific T cells – an off-the-shelf, allogeneic CAR T-cell immunotherapy platform

Background & Aim Chimeric antigen receptor (CAR) T cells have demonstrated potent antitumor efficacy in treating a range of B-cell malignancies, yet technical and commercial challenges using an autologous approach hinder widespread development. Virus-specific T cells present a potential strategy towards an off-the-shelf, allogeneic platform due to the volume of supporting clinical data and additional advantages associated with native TCR engagement to latent virus antigens. Tabelecleucel (tab-cel®) is an investigational off-the-shelf, allogeneic T-cell immunotherapy utilizing native TCR specificity against Epstein-Barr virus (EBV) antigens present in lymphomas and other cancers. With low incidence of GvHD and no cytokine release syndrome, tab-cel® has been shown to be efficacious and well tolerated. Transfer of CAR transgenes into EBV-specific T cells represents an attractive approach to generate off-the-shelf, allogeneic CAR T therapies. Methods, Results & Conclusion We have generated EBV-specific T cells engineered with second generation CD19 CARs as tool compositions to evaluate the potential of this off-the-shelf CAR T approach. Allo-EBV.CD19.CAR T cells express high levels of CD19 CAR and maintain native EBV TCR expression. Allo-EBV.CD19 CAR T cells are predominantly central memory and show CD25, 4-1BB, and IFN-γ activation markers. Functional studies also demonstrate potent antitumor activity against CD19-positive NALM/6 and Raji tumor targets, but no activity against CD19-negative K562 cells. Allo-EBV.CD19.CAR T cells exhibit rapid and comparable killing kinetics compared to CD19.CAR T cells. Allo-CAR T cells also demosntarted antigen-specific proliferation against EBV and CD19-positive targets. Like unmodified EBV-specific T cells, allo.EBV.CD19.CAR T cells maintain the ability to kill B-lymphoblastoid cell lines (BLCL) and little reactivity or inflammatory cytokine secretion in response to HLA mismatched and antigen-negative targets. CD19 CAR engineered EBV T cells demonstrate efficient and potent killing of antigen-positive target cells, while exhibiting little alloreactivity. This platform provides a framework from which we are developing ATA3219, a next generation off-the-shelf, CD19-targeted CAR T utilizing a novel optimized stimulatory domain.

Precise therapeutic gene correction by a simple nuclease-induced double-stranded break.

Current programmable nuclease-based (e.g. CRISPR-Cas9) methods for precise correction of a disease-causing genetic mutation harness the Homology Directed Repair (HDR) pathway. However, this repair process requires co-delivery of an exogenous DNA donor to recode the sequence and can be inefficient in many cell types. Here, we show that disease-causing frameshift mutations resulting from microduplications can be efficiently reverted to the wild-type sequence simply by generating a double-strand break (DSB) near the center of the duplication. We demonstrate this in patient-derived cell lines for two diseases: Limb-Girdle Muscular Dystrophy 2G (LGMD2G)1 and Hermansky-Pudlak Syndrome Type 1 (HPS1)2. Clonal analysis of Streptococcus pyogenes Cas9 (SpyCas9) nuclease-treated LGMD2G iPSCs revealed that ~80% contained at least one wild-type allele and that this correction restored TCAP expression in LGMD2G iPSC-derived myotubes. Efficient genotypic correction was also observed upon SpyCas9 treatment of an HPS1 patient-derived B-lymphoblastoid cell line (B-LCL). Inhibition of PARP-1 (poly (ADP-ribose) polymerase) suppresses the nuclease-mediated collapse of the microduplication to the wild-type sequence, confirming that precise correction is mediated by the MMEJ (microhomology-mediated end joining) pathway. Analysis of editing by SpyCas9 and Lachnospiraceae bacterium ND2006 Cas12a (LbaCas12a) at non-pathogenic microduplications within the genome that range in length from 4 bp to 36 bp indicates that the correction strategy is broadly applicable to a wide range of microduplication lengths and can be initiated by a variety of nucleases. The simplicity, reliability and efficacy of this MMEJ-based therapeutic strategy should permit the development of nuclease-based gene correction therapies for a variety of diseases that are associated with microduplications.

Targeted Therapy for EBV-Associated B-cell Neoplasms.

Epstein-Barr virus (EBV) is directly implicated in several B-cell lymphoid malignancies. EBV-associated lymphomas are characterized by prominent activation of the NF-κB pathway and targeting this pathway establishes a rationale for a therapeutic approach. The ubiquitin/proteasome signaling plays an essential role in the regulation of the NF-κB pathway. Ixazomib is an FDA-approved, orally bioavailable proteasome inhibitor. Here we report the first preclinical evaluation of ixazomib-mediated growth-inhibitory effects on EBV-infected B-lymphoblastoid cell lines Raji and Daudi. Ixazomib induced apoptosis in these cell lines in a dose-dependent manner. Cell-cycle analysis demonstrated ixazomib treatment induced cell-cycle arrest at the G2-M phase with a concomitant decrease in G0-G1 and S phases. The results further revealed an increase in p53, p21, and p27 levels and a decrease in survivin and c-Myc protein levels. Mechanistically, ixazomib treatment resulted in the accumulation of polyubiquitinated proteins, including phosphorylated IκBα with a significant reduction of p65 subunit nuclear translocation. Altogether, our preclinical data support the rationale for in vivo testing of ixazomib in EBV-associated B-cell neoplasms. IMPLICATIONS: This preclinical study supports the use of oral proteasome inhibitor ixazomib for targeting NF-κB signaling in the treatment of EBV-associated B-cell neoplasms.Visual Overview: http://mcr.aacrjournals.org/content/molcanres/17/4/839/F1.large.jpg.

Extensive epigenetic and transcriptomic variability between genetically identical human B-lymphoblastoid cells with implications in pharmacogenomics research

Genotyped human B-lymphoblastoid cell lines (LCLs) are widely used models in mapping quantitative trait loci for chromatin features, gene expression, and drug response. The extent of genotype-independent functional genomic variability of the LCL model, although largely overlooked, may inform association study design. In this study, we use flow cytometry, chromatin immunoprecipitation sequencing and mRNA sequencing to study surface marker patterns, quantify genome-wide chromatin changes (H3K27ac) and transcriptome variability, respectively, among five isogenic LCLs derived from the same individual. Most of the studied LCLs were non-monoclonal and had mature B cell phenotypes. Strikingly, nearly one-fourth of active gene regulatory regions showed significantly variable H3K27ac levels, especially enhancers, among which several were classified as clustered enhancers. Large, contiguous genomic regions showed signs of coordinated activity change. Regulatory differences were mirrored by mRNA expression changes, preferentially affecting hundreds of genes involved in specialized cellular processes including immune and drug response pathways. Differential expression of DPYD, an enzyme involved in 5-fluorouracil (5-FU) catabolism, was associated with variable LCL growth inhibition mediated by 5-FU. The extent of genotype-independent functional genomic variability might highlight the need to revisit study design strategies for LCLs in pharmacogenomics.

Multiple Knockout of Classical HLA Class II β-Chains by CRISPR/Cas9 Genome Editing Driven by a Single Guide RNA.

Comprehensive knockout of HLA class II (HLA-II) β-chain genes is complicated by their high polymorphism. In this study, we developed CRISPR/Cas9 genome editing to simultaneously target HLA-DRB, -DQB1, and -DPB1 through a single guide RNA recognizing a conserved region in exon 2. Abrogation of HLA-II surface expression was achieved in five different HLA-typed, human EBV-transformed B lymphoblastoid cell lines (BLCLs). Next-generation sequencing-based detection confirmed specific genomic insertion/deletion mutations with 99.5% penetrance in sorted cells for all three loci. No alterations were observed in HLA-I genes, the HLA-II peptide editor HLA-DMB, or its antagonist HLA-DOB, showing high on-target specificity. Transfection of full-length HLA-DPB1 mRNA into knockout BLCLs fully restored HLA-DP surface expression and recognition by alloreactive human CD4 T cells. The possibility to generate single HLA-II-expressing BLCLs by one-shot genome editing opens unprecedented opportunities for mechanistically dissecting the interaction of individual HLA variants with the immune system.

Enhancing the antitumor functions of invariant natural killer T cells using a soluble CD1d-CD19 fusion protein

AbstractInvariant natural killer T (iNKT) cells comprise a unique lineage of CD1d-restricted lipid-reactive T lymphocytes that potently kill tumor cells and exhibit robust immunostimulatory functions. Optimal tumor-directed iNKT cell responses often require expression of the antigen-presenting molecule CD1d on tumors; however, many tumor cells downregulate CD1d and thus evade iNKT cell recognition. We generated a soluble bispecific fusion protein designed to direct iNKT cells to the site of B-cell cancers in a tumor antigen-specific but CD1d-independent manner. This fusion protein is composed of a human CD1d molecule joined to a single chain antibody FV fragment specific for CD19, an antigen widely expressed on B-cell cancers. The CD1d-CD19 fusion protein binds specifically to CD19-expressing, but not CD19-negative cells. Once loaded with the iNKT cell lipid agonist α-galactosyl ceramide (αGC), the CD1d-CD19 fusion induces robust in vitro activation of and cytokine production by human iNKT cells. iNKT cells stimulated by the αGC-loaded CD1d-CD19 fusion also strongly transactivate T-, B-, and NK-cell responses and promote dendritic cell maturation. Importantly, the αGC-loaded fusion induces robust lysis of CD19+CD1d− Epstein-Barr virus immortalized human B-lymphoblastoid cell lines that are otherwise resistant to iNKT cell killing. Consistent with these findings; administration of the αGC-loaded fusion protein controlled the growth of CD19+CD1d− tumors in vivo, suggesting that it can “link” iNKT cells and CD19+CD1d− targets in a therapeutically beneficial manner. Taken together, these preclinical studies demonstrate that this B cell–directed fusion protein can be used to effectively induce iNKT cell antitumor responses in vitro and in vivo.

Next-generation HLA typing of 382 International Histocompatibility Working Group reference B-lymphoblastoid cell lines: Report from the 17th International HLA and Immunogenetics Workshop

Extended molecular characterization of HLA genes in the IHWG reference B-lymphoblastoid cell lines (B-LCLs) was one of the major goals for the 17th International HLA and Immunogenetics Workshop (IHIW). Although reference B-LCLs have been examined extensively in previous workshops complete high-resolution typing was not completed for all the classical class I and class II HLA genes. To address this, we conducted a single-blind study where select panels of B-LCL genomic DNA samples were distributed to multiple laboratories for HLA genotyping by next-generation sequencing methods. Identical cell panels comprised of 24 and 346 samples were distributed and typed by at least four laboratories in order to derive accurate consensus HLA genotypes. Overall concordance rates calculated at both 2- and 4-field allele-level resolutions ranged from 90.4% to 100%. Concordance for the class I genes ranged from 91.7 to 100%, whereas concordance for class II genes was variable; the lowest observed at HLA-DRB3 (84.2%). At the maximum allele-resolution 78 B-LCLs were defined as homozygous for all 11 loci. We identified 11 novel exon polymorphisms in the entire cell panel. A comparison of the B-LCLs NGS HLA genotypes with the HLA genotypes catalogued in the IPD-IMGT/HLA Database Cell Repository, revealed an overall allele match at 68.4%. Typing discrepancies between the two datasets were mostly due to the lower-resolution historical typing methods resulting in incomplete HLA genotypes for some samples listed in the IPD-IMGT/HLA Database Cell Repository. Our approach of multiple-laboratory NGS HLA typing of the B-LCLs has provided accurate genotyping data. The data generated by the tremendous collaborative efforts of the 17th IHIW participants is useful for updating the current cell and sequence databases and will be a valuable resource for future studies.

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.

SU105 - MODELING SCHIZOPHRENIA IN VITRO BY THE GENERATION OF NEURONS FROM PATIENT-SPECIFIC STEM CELLS

Background Schizophrenia is a neurodevelopmental disease. Causes include environmental and genetic factors. Characteristics of schizophrenia include a deregulation of the glutamatergic and the GABAergic neurotransmission and the degeneration of predominantly inhibitory interneurons. Recently, 108 loci referring to Single Nucleotide Polymorphisms (SNPs) and 8 loci containing Copy Number Variations (CNVs) have been associated with schizophrenia suggesting a strong impact of genetic factors on the embryonic development of human brain. Together with other research teams, we identified CNVs in the Neurexin 1 gene (NRXN1) in patients suffering from schizophrenia, which highly recommends the detailed analysis of NRXN1-related disease mechanisms. Neurexins are presynaptic transmembrane proteins. We established patient-specific induced Pluripotent Stem (iPS) cells from schizophrenia patients and healthy donors to differentiate neurons for the investigation of disease mechanisms related to NRXN1 in schizophrenia patients. Patient-specific iPS cells carry the disease-associated genetic background of a patient, which is a CNVs affecting NRXN1. Methods Human iPS cells were generated from B-Lymphoblastoid Cell Lines (B-LCLs) using episomal vectors. B-LCLs were obtained from patients carrying CNVs in NRXN1 and healthy donors. The pluripotency of stem cells was verified by alkaline phosphatase staining, immunofluorescence analysis, transcript analysis, western blot analysis, and the induction of three germ layers. A neuronal screening protocol was applied to select iPS cell clones with a high differentiation potential towards neuronal cell lineages. Established lines were differentiated into Neural Stem Cells (NSCs) and further differentiated into functional and mature neurons. Terminal differentiated neurons were characterized by morphology, transcript analysis, western blot analysis, immunofluorescence analysis, electrophysiology, and mitochondrial respiration. Results Transcript analysis and western blot analysis of iPS cells verified permanent induction of pluripotency marker genes including OCT4. The spontaneous differentiation of iPS cells into derivatives of the three germ layers was demonstrated. The induction of neural stem cells was characterized by mRNA and protein analysis of neural stem cells markers including SOX2 and PAX6. Neural induction towards cortical neurons was monitored by a set of neurodevelopmental marker genes including NESTIN, GFAP, and NGN3. The efficient differentiation of mature neurons was shown by the expression of marker genes including glutamate and GABA receptors. Mitochondrial respiration was elevated in differentiated neurons. Patch clamp analysis verified the differentiation of functional GABAergic and glutamatergic neurons. Discussion Human iPS cells provide a powerful tool for the analysis of schizophrenia-associated and patient-specific DNA variations including CNVs. We successfully established mature and functional neuronal cultures suitable to study disease mechanisms related to NRXN1. Accordingly, this approach is an excellent opportunity for the identification of potential therapeutic targets.

SU31 - ESTABLISHING A BLOOD-BRAIN BARRIER MODEL FOR THE ANALYSIS OF ALZHEIMER'S DISEASE USING PATIENT DERIVED INDUCED PLURIPOTENT STEM CELLS

Background Alzheimer's Disease (AD) is the most common form of dementia. One characteristic of AD is the accumulation of amyloid-β (Aβ) peptides in different regions of the brain caused by the sequential cleavage of the transmembrane amyloid precursor protein. These extracellular amyloid plaques are a result of a dysregulated Blood-Brain Barrier (BBB) and emerge either due to an augmented production or a decreased excretion through brain barriers. The BBB with its several transporter systems shows age dependent expression levels of unspecific Aβ transporters. Recently, 19 loci including ABCA7, an ABC transporter responsible for Aβ efflux at the BBB, were discovered showing genome-wide significant association with the sporadic, late-onset form of AD (LOAD). Together with other research groups we identified several mutations in ABCA7 in AD patients, which is why an analysis of mutated ABCA7 in AD-related BBB models should be considered. We established patient-specific pluripotent stem cells from AD patients and healthy donors to differentiate endothelial cells and other related BBB cells for the investigation of disease mechanisms related to ABCA7 in AD patients. Methods Episomal vectors were used for the generation of induced Pluripotent Stem (iPS) cell lines from AD-specific B-lymphoblastoid cell lines (B-LCLs) and control B-LCLs of healthy donors. Alkaline phosphatase staining, immunofluorescence analysis, transcript analysis, western blot analysis, and the induction of three germ layers verified pluripotency. Successfully established lines were applied for the generation of BBB cells and neighboring cells. Endothelial cells and astrocytes were characterized by transcript analysis and flow cytometry. The differentiation process was analyzed for the altered expression of ABCA7. Results AD-specific iPS cells were successfully generated from B-LCLs. We could show alkaline phosphatase activity and the nuclear localization of pluripotency-related transcription factors including OCT4, SOX2, and NANOG. Transcript analysis and western blot analysis verified permanent induction of pluripotency marker genes including OCT4. The spontaneous differentiation of iPS cells into derivatives of the three germ layers was demonstrated. The efficient differentiation of high quality endothelial cells was shown by the expression of endothelial marker genes including von Willebrand factor. Astrocyte-specific markers including EAAT1 demonstrated efficient astrocyte differentiation. Discussion Human iPS cells provide a powerful tool for the analysis of AD-associated and patient-specific mutations in ABCA7. We successfully established endothelial cells and astrocytes representing suitable to study disease mechanisms of the BBB. Accordingly, this approach is an excellent opportunity to analyze pathogenic phenotypes of AD in vitro towards the identification of potential therapeutic targets.

Modeling Human Paroxysmal Nocturnal Hemoglobinuria Via CRISPR/Cas9 HSPC Gene Editing in Non-Human Primate

Recent advances in CRISPR/Cas9 technology allowing precise genome editing at a site of interest have enabled relevant human disease modeling and the development of corrective gene therapies for various genetic disorders. Paroxysmal nocturnal hemoglobinuria (PNH) is a hematological disorder linked to acquired somatic loss-of-function mutations disrupting the X-linked PIG-A gene in hematopoietic stem and progenitor cells (HSPC), characterized by the clinical triad of intravascular hemolysis, thrombosis and bone marrow failure, as well as clonal expansion of HSPC defective in glycosylphophatidylinositol(GPI)-linked proteins, due to loss of PIG-A enzymatic activity. There have been many attempts to model PNH utilizing conditional knockout strategies in mice, but these do not recapitulate the hemolytic phenotype or clonal dominance. Whether clonal expansion is intrinsic to PIG-A deficient HSPC or extrinsic, resulting from relative protection of PIG-A deficient HSPC and progeny cells from immune attack, is unclear. To explore the pathophysiologic mechanisms of PNH HSPC clonal expansion, we generated a relevant non-human primate model for PNH, utilizing autologous transplantation of HSPC edited with CRISPR/Cas9 at the PIG-A locus.The most efficient guide RNAs (gRNAs) targeting several sites within PIG-A exon 2 locus on the X chromosome, where most of mutations occur in patients with PNH, were selected based on efficiency of editing as screened in FRhK-4 cell lines and mobilized macaque CD34+ HSPC. Compared to editing of the control AAVS1 locus and other genes of interest, editing of all PIG-A locus sites was relatively inefficient. Autologous HSPC were transduced with ribonucleotide protein (RNP) complexes of Cas9 protein and selected gRNAs targeting either PIG-A or the AAVS1 “safe harbor” site, as an internal control, followed by transplantation into one female (ZI35) and one male (ZL19) macaque, respectively. Both animals engrafted promptly and clones acquiring the PNH loss-of-function phenotype, defined by loss of binding of FLAER, a fluorescent compound that binds to all GPI anchors, and loss of expression of lineage-specific GPI-linked proteins (CD24 for granulocytes and CD14 for monocytes) were stably maintained at levels of 0.2-0.4% for up to 19 months post-transplantation, with no evidence for intrinsic clonal expansion of PIG-A edited cells (Fig. 1A). Upon targeted deep sequencing, 0.4-1% insertions and deletions (INDELs) induced by CRISPR/Cas9 were detected, and the predominant INDEL type was identified as a single base deletion at the +1 or -1 positions of the target site consistently in granulocytes and other mature hematopoietic lineage cells from peripheral blood (PB), suggesting that initial mutations occurred in long-lasting HSPCs rather than short-term progenitor cells. Furthermore, as the PIG-A gene is located on X chromosome, we sought to investigate the difference in genome editing efficiency depending on the number of X alleles or activation state. Note that the expected INDEL frequency in completely PIG-A deficient sorted FLAERneg cells would be 100%, however, large deletions or rearrangements are not detected by standard deep sequencing methodologies. Interestingly, the mutation frequencies in total granulocytes and more importantly in sorted FLAER negative PNH cells were always much higher in male (ZL19) macaque cells than in female (ZI35) macaque cells (Fig. 1B and C). Consistently, gene editing PIG-A allele efficiency with CRISPR/Cas9 was also higher in human male B-lymphoblastoid cell lines (LCL) compared to female cells, 23.2% versus 16.6% (n=5), respectively. The finding that edited allele frequency was consistently lower in sorted FLAERneg female than male cells suggests that editing of the active X allele may be favored, potentially due to poor accessibility of inactive loci to editing machinery.In conclusion, we have successfully established a rhesus macaque model for PNH utilizing autologous transplantation of CRISPR/Cas9 edited HSPC. To date, we found no evidence for intrinsic expansion of PIG-A deficiency HSPC and hematopoietic progeny. This modeling approach could be utilized for further investigation of extrinsic or intrinsic factor responsible for clonal expansion. Furthermore, our findings provide a better insight into the relationship between CRISPR/Cas9 editing efficiency and active versus inactive X-linked genes. [Display omitted] DisclosuresDunbar:National Institute of Health: Research Funding.

OR20 Investigating the microrna profile of B cell lines for their differential effect on haplotype specific DPB1 gene expression

Aim SNP rs9277534 in the 3’-UTR region of the HLA-DPB1 gene is associated with differential expression of DP molecule on the cell surface of lymphocytes. It appears that this differential expression of DP influences risk of GVHD in unrelated donor hematopoietic cell transplantation and course of hepatitis B infection. We aim to uncover mechanisms for such alterations in protein expression by identifying haplotype-specific microRNAs (miRNAs) and their differential in silico targeting patterns that occur at polymorphic sites, with eventual experimental confirmation. Methods Novel and miRBase-annotated miRNAs expressed in COX (high DP expression) and PGF (low DP expression) B-lymphoblastoid cell lines (BLCL) were characterized using miRDeep2. Higher-confidence miRNAs were obtained by requiring > 80% estimated true positive probability, RANDFOLD p ⩽ 0.05, and removal of entries overlapping non-miRNA Rfam elements. A miRNA target prediction scheme utilizing complementary TargetScan v7 (TS) and RNA22v2 (R22) was employed, using the following relatively stringent parameter settings to identify the highest-precision targets: context++ score Results COX and PGF BLCLs demonstrated differential miRNA expression and predicted targeting at polymorphic sites of their respective DPB1 genes. Differential predicted targets were identified in exon 2, introns 1, 2, and 3 and the 3’-UTR of the two DPB1 sequences. These identified miRNAs have the potential to explain altered protein expression levels via putative cytoplasmic or nuclear miRNA effects. rs9277534 is not included among the high-confidence targeted sites; however, if thresholds are relaxed, then that region is differentially targeted as well. Conclusions We have identified miRNAs with differential expression in COX and PGF BLCLs, which demonstrate unique predicted targeting patterns at polymorphic sites of their respective HLA-DPB1 alleles. This catalog of miRNAs can now be tested in further experiments to identify true mediators of HLA-DPB1 expression and help to identify actual causative polymorphisms in such regions that involve extensive and confounding linkage disequilibrium.

An allelic variant in the intergenic region between ERAP1 and ERAP2 correlates with an inverse expression of the two genes

The Endoplasmatic Reticulum Aminopeptidases ERAP1 and ERAP2 are implicated in a variety of immune and non-immune functions. Most studies however have focused on their role in shaping the HLA class I peptidome by trimming peptides to the optimal size. Genome Wide Association Studies highlighted non-synonymous polymorphisms in their coding regions as associated with several immune mediated diseases. The two genes lie contiguous and oppositely oriented on the 5q15 chromosomal region. Very little is known about the transcriptional regulation and the quantitative variations of these enzymes. Here, we correlated the level of transcripts and proteins of the two aminopeptidases in B-lymphoblastoid cell lines from 44 donors harbouring allelic variants in the intergenic region between ERAP1 and ERAP2. We found that the presence of a G instead of an A at SNP rs75862629 in the ERAP2 gene promoter strongly influences the expression of the two ERAPs with a down-modulation of ERAP2 coupled with a significant higher expression of ERAP1. We therefore show here for the first time a coordinated quantitative regulation of the two ERAP genes, which can be relevant for the setting of specific therapeutic approaches.

Protective C allele of the single-nucleotide polymorphism rs1335532 is associated with strong binding of Ascl2 transcription factor and elevated CD58 expression in B-cells

CD58 is expressed on the surface of antigen-presenting cells, including B-cells, and provides co-stimulation to regulatory T-cells (Treg) through CD2 receptor binding. Tregs appear to be essential suppressors of tissue-specific autoimmune responses. Thereby, CD58 plays protective role in multiple sclerosis (MS) and CD58 was identified among several loci associated with MS susceptibility. Minor (C) variant of the single-nucleotide polymorphism (SNP) rs1335532 is associated with lower MS risk according to genome-wide association studies (GWAS) and its presence correlates with higher CD58 mRNA levels in MS patients. We found that genomic region containing rs1335532 has enhancer properties and can significantly boost the CD58 promoter activity in lymphoblast cells. Using bioinformatics and pull-down assay we found that the protective (C) rs1335532 allele created functional binding site for ASCL2 transcription factor, a target of the Wnt signaling pathway. Both in B-lymphoblastoid cell lines and in primary B-cells, as well as in a monocytic cell line, activation of Wnt signaling resulted in an increased CD58 promoter activity in the presence of the protective but not the risk allele of rs1335532, whereas ASCL2 knockdown abrogated this effect. In summary, our results suggest that ASCL2 mediates the protective function of rs1335532 minor (C) allele in MS.

Identification of Two Lpp20 CD4+ T Cell Epitopes in Helicobacter pylori-Infected Subjects.

Antigen-specific CD4+ T cells play an essential role in effective immunity against Helicobacter pylori (H. pylori) infection. Lpp20, a conserved lipoprotein of H. pylori, has been investigated as one of major protective antigens for vaccination strategies. Our previous study identified two H-2d-restricted CD4+ T cell epitopes within Lpp20 and an epitope vaccine based on these epitopes was constructed, which protected mice in prophylactic and therapeutic vaccination against H. pylori infection. Immunodominant CD4+ T cell response is an important feature of antiviral, antibacterial, and antitumor cellular immunity. However, while many immunodominant HLA-restricted CD4+ T cell epitopes of H. pylori protective antigens have been identified, immunodominant HLA-restricted Lpp20 CD4+ T cell epitope has not been elucidated. In this study, a systematic method was used to comprehensively evaluate the immunodominant Lpp20-specific CD4+ T cell response in H. pylori-infected patients. Using in vitro recombinant Lpp20 (rLpp20)-specific expanded T cell lines from H. pylori-infected subjects and 27 18mer overlapping synthetic peptides spanned the whole Lpp20 protein, we have shown that L55–72 and L79–96 harbored dominant epitopes for CD4+ T cell responses. Then the core sequence within these two 18mer dominant epitopes was screened by various extended or truncated 13mer peptides. The immunodominant epitope was mapped to L57–69 and L83–95. Various Epstein-Barr virus (EBV) transformed B lymphoblastoid cell lines (B-LCLs) with different HLA alleles were used as antigen presenting cell (APC) to present peptides to CD4+ T cells. The restriction molecules were determined by HLA class-antibody blocking. L57–69 was restricted by DRB1-1501 and L83–95 by DRB1-1602. The epitopes were recognized on autologous dendritic cells (DCs) loaded with rLpp20 but also those pulsed with whole cell lysates of H. pylori (HP-WCL), suggesting that these epitopes are naturally processed and presented by APC. CD4+ T cells were isolated from H. pylori-infected patients and stimulated with L57–69 and L83–95. These two epitopes were able to stimulate CD4+ T cell proliferation. This study may be of value for the future development of potential H. pylori vaccine.

Short Tandem Repeat analysis after Whole Genome Amplification of single B-lymphoblastoid cells

To allow multiple genetic analyses on a single cell, whole genome amplification (WGA) is required. Unfortunately, studies comparing different WGA methods for downstream human identification Short Tandem Repeat (STR) analysis remain absent. Therefore, the aim of this work was to assess the performance of four commercially available WGA kits for downstream human identification STR profiling on a B-lymphoblastoid cell line. The performance was assessed using an input of one or three micromanipulated cells. REPLI-g showed a very low dropout rate, as it was the only WGA method in this study that could provide a complete STR profile in some of its samples. Although Ampli1, DOPlify and PicoPLEX did not detect all selected STR markers, they seem suitable for genetic identification in single-cell applications.

Modulating Gene Expression in Epstein-Barr Virus (EBV)-Positive B Cell Lines with CRISPRa and CRISPRi.

Epstein-Barr virus (EBV) transforms small resting primary B cells into large lymphoblastoid cells which are able to grow and survive in vitro indefinitely. These cells represent a model for oncogenesis. In this unit, variants of conventional clustered regularly interspaced short palindromic repeats (CRISPR), namely the CRISPR activation (CRISPRa) and CRISPR interference (CRISPRi) methods, are discussed in the context of gene regulation at genomic DNA promoter and enhancer elements. Lymphoblastoid B cell lines (LCLs) stably expressing nuclease-deficient Cas9 (dCas9)-VP64 (Cas9 associated with CRISPRa) or dCas9-KRAB (Cas9 associated with CRISPRi) are transduced with lentivirus that encodes a single guide RNA (sgRNA) that targets a specific gene locus. The ribonucleoprotein complex formed by the dCas9 molecule and its cognate sgRNA enables sequence-specific binding at a promoter or enhancer of interest to affect the expression of genes regulated by the targeted promoter or enhancer. © 2018 by John Wiley & Sons, Inc.