Multiple Healthy Donors Research Articles

Novel Long Non Coding RNA Blackmamba Is Associated to ALK- anaplastic Large Cell Lymphoma

Systemic Anaplastic Large Cell Lymphomas (ALCLs) is among the common subtypes of T-cell lymphomas. The prognosis of ALCLs patients has been proven to be associated with translocations of the anaplastic lymphoma kinase (ALK) gene, having ALK negative patients (ALK-ALCL) a poorer 5-years survival rate (30-40%). We now recognize that ALK- ALCL includes multiple subsets some of which are bear unique genetic defects and unique clinical outcomes. Long non coding RNAs (lncRNAs) are regulators of gene expression controlling critical processes (epigenetic regulation, imprinting, cell cycle, apoptosis) frequently deregulated in cancer. In T-cells, more than 500 lncRNAs are associated with lymphocyte signatures, control cell differentiation and cell identity. While lncRNAs are found to be differentially expressed in solid cancer and directly linked to the different stages of carcinogenesis, their role in lymphoid neoplasms is largely unknown.To identify novel lncRNAs maintaining the neoplastic phenotype of ALCL, we prepared cDNA libraries representative of coding and non-coding RNA. High coverage and directional RNA-sequencing was performed in 30 purified human T lymphocytes from multiple healthy donors, corresponding to different stages of differentiation, 22ALK+ ALCL and 20 ALK-ALCL patients' samples. Seven bona fide ALCL cell lines were included.By de novo transcriptome reconstruction and using a new bioinformatic pipeline, we identified 83lncRNAs exclusively expressed in ALCL patient's samples. Among them, 82 lncRNAs were coshared between ALK+ and ALK- ALCL and only one was exclusively expressed in ALK-ALCL samples. We named this new lncRNA BlackMamba.We selected the 10 top-scoring ALCL-associated lncRNAs and BlackMamba and determined their expression by RT-PCR, in a validation set of ALCL (ALK+: n=3, ALK-: n=3), T-ALL (n=8), PTCL (n=9), and AITL (n=9) samples. Resting and activated PBMCs from two additional donors were included.Our data confirmed that the 10 top-scoring ALCL-associated lncRNAs were expressed exclusively in ALCLs without differences in the expression among ALCL subtypes. Moreover, BlackMamba was detected only in ALCL samples with a preferential expression in ALK-ALCL samples. All lncRNAs were not expressed in normal T-lymphocytes.We next determine the BlackMamba expression in ALCL (n=6), T-ALL (n=3), cutaneous T-lymphomas (n=3) cell lines by RT-PCR. FedP, MAC1 and MAC2a (ALK-ALCL) cell lines showed the highest expression.Because the cellular localization of lncRNAs affects their function, we assessed the sub-cellular localization of BlackMamba by nucleo/cytoplasm cell fractionation and RT-PCR in FedP and MAC2A cell lines. BlackMamba displayed a preferential nuclear/chromatin-associated localization. Having proven that JAK/STAT signaling pathway plays a key role in both ALK+ and ALK-ALCL, we test whether the expression of BlackMamba could be modulated after treatment with JAK1/2 (ruxolitinib) TKi. Our data showed that, in FedP and MAC2A cell lines, BlackMamba expression was 50% down-regulated relative to control.Next, to identify whether BlackMamba may have a unique associated to specific chromatin remodel proteins, we verified its association with several candidates and found the physical interaction with the histone-lysine N-methyltransferase enzyme EZH2 by RIP in the FedP cells.Notably, after BlackMamba knock down, the proliferation of FedP cells was reproducibly reduced (25% at 84hrs using multiple RNAi oligonucleotides).Collective, our data indicate that ALCL aberrantly express novel and uncharacterized lncRNAs and that BlackMamba is a novel lncRNA associated with ALK-ALCL. Moreover, BlackMamba may contributes to the maintenance of ALK-ALCL neoplastic phenotype. DisclosuresMerli:Roche: Membership on an entity's Board of Directors or advisory committees, Research Funding; Teva Pharmaceuticals Industries: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees.

Transcriptome profiling of human FoxP3+ regulatory T cells

The major goal of this study was to perform an in depth characterization of the “gene signature” of human FoxP3+ T regulatory cells (Tregs). Highly purified Tregs and T conventional cells (Tconvs) from multiple healthy donors (HD), either freshly explanted or activated in vitro, were analyzed via RNA sequencing (RNA-seq) and gene expression changes validated using the nCounter system. Additionally, we analyzed microRNA (miRNA) expression using TaqMan low-density arrays. Our results confirm previous studies demonstrating selective gene expression of FoxP3, IKZF2, and CTLA4 in Tregs. Notably, a number of yet uncharacterized genes (RTKN2, LAYN, UTS2, CSF2RB, TRIB1, F5, CECAM4, CD70, ENC1 and NKG7) were identified and validated as being differentially expressed in human Tregs. We further characterize the functional roles of RTKN2 and LAYN by analyzing their roles in vitro human Treg suppression assays by knocking them down in Tregs and overexpressing them in Tconvs. In order to facilitate a better understanding of the human Treg gene expression signature, we have generated from our results a hypothetical interactome of genes and miRNAs in Tregs and Tconvs.

Ibrutinib Enhances the Activity of Anti-CD20 Antibodies in an MCL Mouse Model: Effect of Drug at Clinically Relevant Concentrations on ADCC and ADCP

Introduction: Ibrutinib, a covalent inhibitor of Bruton's tyrosine kinase (BTK), is clinically established as an effective treatment for chronic lymphocytic leukemia (CLL) and mantle cell lymphoma (MCL). Further, recent trials reported that ibrutinib combined with anti-CD20 monoclonal antibodies (CD20 mAbs) led to overall response rates (ORRs) of 95% and 100% in relapsed CLL patients (Burger et al Lancet Oncol 2014, Jaglowski et al Blood 2015), and an ORR of 87% in relapsed MCL patients (Wang et al ASH 2014), indicating complementary therapeutic effects from the 2 drugs. However, some preclinical studies showed that ibrutinib inhibited antibody-dependent cell-mediated cytotoxicity and phagocytosis (ADCC and ADCP). This discrepancy may be due to differences between experimental conditions (eg, drugs used at micromolar concentrations for sustained periods of time) relative to the pharmacokinetics (PK) of ibrutinib, which has a typical peak plasma concentration (Cmax) of ~200 nM 2 hours (tmax) after oral administration of 420-560 mg, and is cleared rapidly from circulation (Davis et al ASH 2014; Advani et al J Clin Oncol 2013). Using clinical PK as a basis, we designed in vitro and in vivo studies to re-evaluate the impact of ibrutinib on ADCC and ADCP.Methods: To model the effect of covalent inhibition by ibrutinib long after tmax, peripheral blood mononuclear cells (PBMC) from multiple healthy donors were incubated for 2 hrs with 200 nM ibrutinib, washed, and rested for 12 hours to represent a time halfway to the next daily dose. Natural killer (NK) cells purified from these PBMC were cultured with JeKo-1 (MCL line) or primary CLL cells opsonized with the CD20 mAbs rituximab (RTX), ofatumumab (OFA), or obinutuzumab (OBZ; washout protocol). To assess the effect of ibrutinib at tmax, treated PBMC, without being washed or rested, were used to purify NK cells for an ADCC assay in the presence of 200 nM ibrutinib (no wash protocol). Dead target tumor cells were counted by flow cytometry and presented as % maximum killing by NK cells from PBMC treated with vehicle. In ADCP assays, % PBMC-derived macrophages that had phagocytosed target cells are shown. In a xenograft model of MCL, SCID mice (10/group) bearing subcutaneous Mino tumors were treated with CD20 mAbs with or without daily oral dosing with ibrutinib at 12 mg/kg. Tumor size was recorded twice weekly. Plasma concentration of ibrutinib was measured using LC-MS. BTK occupancy in tumor cells by ibrutinib was determined as described previously (Dubovsky et al Blood 2013).Results: The ADCC activity of NK cells treated with ibrutinib rebounded significantly after the washout procedure. Killing of RTX-opsonized JeKo-1 and primary CLL cells rose from 36% and 21% (no wash) to 77% and 71% (wash), respectively. Similar results were obtained using OFA. The killing of OBZ-opsonized JeKo-1 cells was not substantially affected with or without washouts (Fig 1A). Phagocytosis was not affected in the presence of 200 nM ibrutinib (Fig 1B). In tumor-bearing mice, ibrutinib administered at 12 mg/kg by oral gavage resulted in a Cmax of 338 ng/mL (767 nM), exceeding the Cmax observed in patients receiving up to 560 mg a day. However, total drug exposure over time (AUC0-last = 391 ng·h/mL) was within range (Advani et al J Clin Oncol 2013, de Jong et al Cancer Chem Pharmacol 2015). In contrast to previous preclinical study results, ibrutinib significantly enhanced the therapeutic effect of CD20 mAbs (Fig 2). At study termination, we confirmed 98.4 ± 0.3% BTK occupancy in tumor cells.Conclusions: We recommend ibrutinib concentrations used in cellular assays and in vivo models reflect pharmacologic levels of ibrutinib in patients administered 420-560 mg once daily. The optimal dose in animal studies can be determined by PK/PD measurements. Our data indicate that NK-mediated ADCC recovers significantly 12 hours after ibrutinib has been removed, which may be attributed to turnover of ITK, or other signaling components of ADCC that are targets of ibrutinib during the rest period. ADCP activity of macrophages was not affected by 200 nM ibrutinib. These data suggest that the 2 major effector cell types for tumor-targeting antibodies are not as inhibited by clinically relevant concentrations of ibrutinib as have been described previously. This is supported by the present in vivo study data showing that ibrutinib did not inhibit, but augmented the therapeutic effect of CD20 mAbs. [Display omitted] [Display omitted] DisclosuresNg:Pharmacyclics LLC, an AbbVie Company: Employment. Lu:Pharmacyclics LLC, an AbbVie Company: Employment. Sukbuntherng:Pharmacyclics LLC, an AbbVie Company: Employment. Neuenburg:Pharmacyclics LLC, an AbbVie Company: Employment. James:Pharmacyclics LLC, an AbbVie Company: Employment. Chang:Pharmacyclics LLC, an AbbVie Company: Employment.

Carbon nanotube-bilirubin oxidase bioconjugate as a new biofuel cell label for self-powered immunosensor.

We demonstrated a biofuel cells (BFCs)-based self-powered sensing system for the detection of Nε-(carboxymethyl)lysine (CML), in which the bilirubin oxidase (BOD)-carbon nanotube (CNT) bioconjugate modified with antibody acted as a biocatalyst for enhancing O2 reduction in the biocathode, as well as the transducing enzyme for signaling magnification. With an increase in the concentration of CML, the amount of BOD labels on biocathode surface increases, thus leading to the higher output of the as-prepared BFCs. This novel BFCs-based self-powered sensor showed a wide linear range for analyzing CML from 1 nM to 100 μM with a detection limit of 0.2 nM, which was 50 times more sensitive than that determined from the conventional ELISA. Most importantly, our new self-powered sensing platform can determine the level of CML in serum samples from multiple healthy donors and multiple sclerosis patients, being well in accordance with that from the commercial ELISA analysis.

A Bayesian hierarchical method to account for random effects in cytogenetic dosimetry based on calibration curves

The dicentric chromosome assay (DCA) is one of the most sensitive and reliable methods of inferring doses of radiation exposure in patients. In DCA, one calibration curve is prepared in advance by in vitro irradiation to blood samples from one or sometimes multiple healthy donors in considering possible inter-individual variability. Although the standard method has been demonstrated to be quite accurate for actual dose estimates, it cannot account for random effects, which come from such as the blood donor used to prepare the calibration curve, the radiation-exposed patient, and the examiners. To date, it is unknown how these random effects impact on the standard method of dose estimation. We propose a novel Bayesian hierarchical method that incorporates random effects into the dose estimation. To demonstrate dose estimation by the proposed method and to assess the impact of inter-individual variability in samples from multiple donors on the estimation, peripheral blood samples from 13 occupationally non-exposed, non-smoking, healthy individuals were collected and irradiated with gamma rays. The results clearly showed significant inter-individual variability and the standard method using a sample from a single donor gave anti-conservative confidence interval of the irradiated dose. In contrast, the Bayesian credible interval for irradiated dose calculated by the proposed method using samples from multiple donors properly covered the actual doses. Although the classical confidence interval of calibration curve with accounting inter-individual variability in samples from multiple donors was roughly coincident with the Bayesian credible interval, the proposed method has better reasoning and potential for extensions.

Impairment of viral replication capacity by nef alleles from HIV elite controllers

Background Elite controllers (EC) are HIV-1 infected persons who can spontaneously control viremia to extremely low levels. Because it is known that nef is required for HIV1 replication and pathogenesis in vivo, we wish to clarify how HIV-1 nef plays a role in acquiring EC phenotype. Materials and methods Viral RNAs were extracted from plasma samples of 45 ECs and 48 chronic progressors (CP) and used for amplification of nef by RT-PCR. The amplified products were cloned into a plasmid and subsequently sequenced. A representative nef clone was selected for each patient and cloned into an NL43 proviral plasmid backbone. Infectious recombinant viruses were prepared by transfecting 293T cells with these resultant proviral clones and tested for their viral replication capacity in PBMCs obtained from multiple healthy donors. Viral replication was monitored by measuring the concentration of p24 Gag in the culture supernatant by p24 ELISA every 3 days. The same virus stocks were further tested for their infectivity using TZM-bl cells. Results All nef alleles isolated from EC and CP had intact ORFs. Phylogenetic analysis revealed no specific lineage or clustering, suggesting the absence of common genetic defects in EC-derived nef alleles. All viruses expressing patient-derived nef showed a wide range of viral replication in PBMC, confirming the importance of nef alleles in the enhancement of viral replication capacity. The viruses harboring EC-derived nef replicated and peaked on Day 12 after infection or even later; whereas the viruses harboring CP-derived nef peaked on Day 9. Also, the peak level of p24 values was significantly lower in the EC-nef viruses than in the CP-nef viruses. Because Nef activity is highly dependent on PBMC donors, we tested 4 different donors’ PBMC and compared the initial burst of their viral replication at Day 9. In all donors’PBMC, viruses carrying EC-nef displayed lower p24 values than those carrying CP-nef, suggesting that the viral replication capacity was much impaired by nef alleles in EC. Corroboratively, in viral infectivity assay in TZM-bl cells, the EC-nef viruses showed significantly lower infectivity than the CP-nef viruses. Conclusion

In vitro human CD4+ T cell response to the vaccinia protective antigens B5R and A33R

Subunit vaccine candidates against poxvirus infection induced protective humoral and cellular response in animal models but their immunogenicity in human remains unknown. We have therefore evaluated in vitro the CD4 T cell response of the major antigens B5R and A33R and characterized their CD4 T cell epitopes. Twelve peptides selected on the basis of their binding capacity to HLA-DR molecules, induced CD4 T lymphocytes harvested in healthy donors. In the A33R proteins two peptides are T cell stimulating for at least half of the donors and are restricted to multiple HLA-DR molecules in agreement with their broad specificity for HLA-DR molecules. In B5R, two peptides exhibited a good immunoprevalence but only one is a good binder to multiple HLA-DR molecules. One peptide was a moderate binder for multiple HLA-DR molecules, although it was efficiently presented to peptide-specific T cell lines. Altogether, our data demonstrated the capacity of B5R and A33R peptides to elicit a T cell response in multiple healthy donors and showed that promiscuity and immunoprevalence of CD4 T cell epitopes are not necessarily associated.