Primary Lymphoid Cells Research Articles

RNA editing by ADAR1 regulates innate and antiviral immune functions in primary macrophages

ADAR1-dependent A-to-I editing has recently been recognized as a key process for marking dsRNA as self, therefore, preventing innate immune activation and affecting the development and resolution of immune-mediated diseases and infections. Here, we have determined the role of ADAR1 as a regulator of innate immune activation and modifier of viral susceptibility in primary myeloid and lymphoid cells. We show that ADAR1 knockdown significantly enhanced interferon, cytokine and chemokine production in primary macrophages that function as antiviral paracrine factors, rendering them resistant to HIV-1 infection. ADAR1 knockdown induced deregulation of the RLRs-MAVS signaling pathway, by increasing MDA5, RIG-I, IRF7 and phospho-STAT1 expression, an effect that was partially rescued by pharmacological blockade of the pathway. In summary, our results demonstrate a role of ADAR1 in regulating innate immune function in primary macrophages, suggesting that macrophages may play an essential role in disease associated to ADAR1 dysfunction. We also show that viral inhibition is exclusively dependent on innate immune activation consequence of ADAR1 knockdown, pointing towards ADAR1 as a potential target to boost antiviral immune response.

The prohibitin protein complex promotes mitochondrial stabilization and cell survival in hematologic malignancies.

Prohibitins (PHB1 and PHB2) have been proposed to play important roles in cancer development and progression, however their oncogenic mechanism of action has not been fully elucidated. Previously, we showed that the PHB1 and PHB2 protein complex is required for mitochondrial homeostasis and survival of normal human lymphocytes. In this study, novel evidence is provided that indicates mitochondrial prohibitins are overexpressed in hematologic tumor cells and promote cell survival under conditions of oxidative stress. Immunofluorescent confocal microscopy revealed both proteins to be primarily confined to mitochondria in primary patient lymphoid and myeloid tumor cells and tumor cell lines, including Kit225 cells. Subsequently, siRNA-mediated knockdown of PHB1 and PHB2 in Kit225 cells significantly enhanced sensitivity to H2O2-induced cell death, suggesting a protective or anti-apoptotic function in hematologic malignancies. Indeed, PHB1 and PHB2 protein levels were significantly higher in tumor cells isolated from leukemia and lymphoma patients compared to PBMCs from healthy donors. These findings suggest that PHB1 and PHB2 are upregulated during tumorigenesis to maintain mitochondrial integrity and therefore may serve as novel biomarkers and molecular targets for therapeutic intervention in certain types of hematologic malignancies.

Epithelial chemokine CXCL14 synergizes with CXCL12 via allosteric modulation of CXCR4.

The chemokine receptor, CXC chemokine receptor 4 (CXCR4), is selective for CXC chemokine ligand 12 (CXCL12), is broadly expressed in blood and tissue cells, and is essential during embryogenesis and hematopoiesis. CXCL14 is a homeostatic chemokine with unknown receptor selectivity and preferential expression in peripheral tissues. Here, we demonstrate that CXCL14 synergized with CXCL12 in the induction of chemokine responses in primary human lymphoid cells and cell lines that express CXCR4. Combining subactive concentrations of CXCL12 with 100–300 nM CXCL14 resulted in chemotaxis responses that exceeded maximal responses that were obtained with CXCL12 alone. CXCL14 did not activate CXCR4-expressing cells (i.e., failed to trigger chemotaxis and Ca2+ mobilization, as well as signaling via ERK1/2 and the small GTPase Rac1); however, CXCL14 bound to CXCR4 with high affinity, induced redistribution of cell-surface CXCR4, and enhanced HIV-1 infection by >3-fold. We postulate that CXCL14 is a positive allosteric modulator of CXCR4 that enhances the potency of CXCR4 ligands. Our findings provide new insights that will inform the development of novel therapeutics that target CXCR4 in a range of diseases, including cancer, autoimmunity, and HIV.—Collins, P. J., McCully, M. L., Martínez-Muñoz, L., Santiago, C., Wheeldon, J., Caucheteux, S., Thelen, S., Cecchinato, V., Laufer, J. M., Purvanov, V., Monneau, Y. R., Lortat-Jacob, H., Legler, D. F., Uguccioni, M., Thelen, M., Piguet, V., Mellado, M., Moser, B. Epithelial chemokine CXCL14 synergizes with CXCL12 via allosteric modulation of CXCR4.

Abstract A83: Hexavalent agonists targeting co-stimulatory receptors of the tumor necrosis factor superfamily

Abstract Tumor necrosis factor receptor superfamily (TNFRSF) proteins are widely expressed by immune and tumor cells highlighting their importance in multiple locations and phases of the anti-tumor immune response. Whereas agonistic stimulation of TRAIL-Receptor-1 or -2 can directly induce apoptosis in tumor cells, signaling through many TNFRSF members, such as CD40, CD27, OX40, HVEM, GITR and 4-1BB, results in co-stimulation of immune cells. Therefore, agonistic stimulation of certain members of the TNFRSF is considered to have a positive impact on immune-based therapeutic concepts in clinical oncology. Apogenix has developed a proprietary technology platform for the construction of novel hexavalent TNFRSF agonists (HERA) for the treatment of cancer. HERA fusion proteins are based on a perfect molecular mimic of the TNFSF cytokine structure. The HERA core unit consists of one single polypeptide chain comprising the three receptor-binding domain-forming subsequences (TNFSF-protomers). These single-chain TNFSF receptor-binding domains (scTNFSF-RBD) preserve the structural organization of the trimeric natural TNFSF cytokine and can be utilized to engineer fully human fusion-proteins in a modular manner. Fusing an IgG1 Fc-domain as a dimerization scaffold to the C-terminus of a scTNFSF-RBD creates a hexavalent agonist from two trivalent scTNFSF-RBDs. As a result of this molecular design, each molecule is capable of clustering six receptors in a spatially well-defined manner in close proximity to each other. Therefore, signaling following treatment with the Apogenix HERA scTNFSF-RBD-Fc fusion proteins in vivo is independent of secondary clustering through Fc-γ receptors that is required for many agonistic anti-TNFRSF antibodies (e.g., anti-TRAIL-R2 or anti-CD40). The described HERA engineering concept has been successfully translated to TRAIL, CD40L, LIGHT and CD27L resulting in hexavalent agonists suitable for further development. CD27-Ligand is a potent co-stimulatory molecule that drives T-cell activation and survival through interaction with its receptor (CD27). Here we show in vitro and in vivo data for APG1293 (scCD27L-RBD-Fc), a hexavalent CD27 agonist. APG1293 was expressed in CHO suspension cells followed by a lab-scale purification process including affinity chromatography and SEC-based polishing, resulting in homogenous aggregate-free protein lots. The purified protein binds its respective target-receptor with high affinity. In vivo stability/PK studies have been performed in addition to in vitro experiments with primary human and mouse lymphoid and myeloid cell populations. Specifically, APG1293 was able to bind CD27 expressed on primary human CD4+ and CD8+ T cells and importantly, binding significantly increased T-cell expansion following activation. In vivo the efficacy of APG1293 was evaluated in the colorectal syngeneic murine tumor models MC38-CEA and CT26. In both models APG1293 treatment resulted in a dose dependent tumor growth inhibition. In summary, the data on the hexavalent APG1293 indicate a potent immune cell driven anti tumor efficacy. Therefore, APG1293 could be applied as a single agent or in combination with check-point inhibitors. Citation Format: Christian Gieffers, Christian Merz, David Richards, Mauricio Redondo, Viola Marschall, Jaromir Sykora, Meinolf Thiemann, Harald Fricke, Oliver Hill. Hexavalent agonists targeting co-stimulatory receptors of the tumor necrosis factor superfamily. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2016 Oct 20-23; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2017;5(3 Suppl):Abstract nr A83.

PARP Inhibitors in Clinical Use Induce Genomic Instability in Normal Human Cells.

Poly(ADP-ribose) polymerases (PARPs) are the first proteins involved in cellular DNA repair pathways to be targeted by specific inhibitors for clinical benefit. Tumors harboring genetic defects in homologous recombination (HR), a DNA double-strand break (DSB) repair pathway, are hypersensitive to PARP inhibitors (PARPi). Early phase clinical trials with PARPi have been promising in patients with advanced BRCA1 or BRCA2-associated breast, ovary and prostate cancer and have led to limited approval for treatment of BRCA-deficient ovary cancer. Unlike HR-defective cells, HR-proficient cells manifest very low cytotoxicity when exposed to PARPi, although they mount a DNA damage response. However, the genotoxic effects on normal human cells when agents including PARPi disturb proficient cellular repair processes have not been substantially investigated. We quantified cytogenetic alterations of human cells, including primary lymphoid cells and non-tumorigenic and tumorigenic epithelial cell lines, exposed to PARPi at clinically relevant doses by both sister chromatid exchange (SCE) assays and chromosome spreading. As expected, both olaparib and veliparib effectively inhibited poly-ADP-ribosylation (PAR), and caused marked hypersensitivity in HR-deficient cells. Significant dose-dependent increases in SCEs were observed in normal and non-tumorigenic cells with minimal residual PAR activity. Clinically relevant doses of the FDA-approved olaparib led to a marked increase of SCEs (5-10-fold) and chromatid aberrations (2-6-fold). Furthermore, olaparib potentiated SCE induction by cisplatin in normal human cells. Our data have important implications for therapies with regard to sustained genotoxicity to normal cells. Genomic instability arising from PARPi warrants consideration, especially if these agents will be used in people with early stage cancers, in prevention strategies or for non-oncologic indications.

Abstract 1204: A construction platform for hexavalent agonists targeting receptors of the tumor necrosis factor superfamily: Where death meets co-stimulation

Abstract Tumor necrosis factor receptor superfamily (TNFRSF) proteins are widely expressed by immune and tumor cells. Their importance in many locations and phases of the anti-tumor immune response is now broadly appreciated and several TNFR agonists are currently in preclinical and clinical development. Importantly, signaling through many TNFRSF members, such as CD40, CD27, OX40, 4-1BB, HVEM and GITR, is potentially associated with an enhanced anti tumor response via co-stimulation of immune cells. Apogenix has established a development platform for a novel class of TNFRSF-agonists for the treatment of cancer. Unlike their natural homotrimeric counterparts, the Apogenix recombinant TNFSF proteins consist of one single polypeptide chain composed of three receptor-binding domain-forming protomers. These single-chain TNFSF receptor-binding domains (scTNFSF-RBD) are mimics of the three-dimensional organization of the natural TNFSF-cytokine and can be used to engineer fully human fusion-proteins from a modular toolbox. For example, fusing an IgG1 Fc-domain to the C-terminus of a scTNFSF-RBD creates a hexavalent agonist as the Fc-domain acts as a dimerization scaffold for two trivalent scTNFSF-RBDs. As a result of this molecular design, each drug molecule is capable of clustering six receptors in a spatially well-defined manner. Consequently, TNFSF receptor signaling following treatment with the Apogenix scTNFSF-RBD-Fc in vivo is independent of secondary clustering through Fc-γ receptors that is required for many anti-TNFRSF agonistic antibodies (e.g., anti-TRAILR2 or -CD40). Following up the scTRAIL-RBD-Fc prototype, this engineering concept has now been successfully translated to CD40L and CD27L resulting in hexavalent agonists suitable for further development. Expression of the drug candidates in CHO suspension cells followed by an AFC and SEC-based lab-scale purification process resulted in homogenous aggregate-free protein lots. The purified proteins bind their respective target-receptors with high affinity. In vivo stability/PK studies have been performed in addition to in vitro experiments with primary human and mouse lymphoid and myeloid cell populations. Specifically, it was shown that scCD27L-RBD-Fc was able to bind CD27 expressed on primary human CD4+ and CD8+ T cells. Importantly, binding significantly increased T cell expansion following activation. Treatment with scCD40L-RBD-Fc induced differentiation of B cells and enhanced primary human monocyte differentiation into DCs or M1 macrophages. Encouraged by the promising results obtained with TRAIL, CD40L, and CD27L, Apogenix is currently expanding the TNFRSF-agonist pipeline to target additional cell populations, locations and phases of the immune response in order to develop novel therapies to treat cancer and other conditions. Citation Format: Christian Merz, Christian Gieffers, Michael Kluge, David M. Richards, Tim Schnyder, Jaromir Sykora, Meinolf Thiemann, Harald Fricke, Oliver Hill. A construction platform for hexavalent agonists targeting receptors of the tumor necrosis factor superfamily: Where death meets co-stimulation. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1204.

Development of pseudotyped retroviral system for effective gene transfer and expression in penaeid shrimp cells

The pantropic retroviral expression system has been widely used to stably introduce foreign genes into the genome of dividing cells from non-mammalian systems. However, we found that the current commercial retrovirus expression system did not work in inactively dividing primary shrimp cells. To overcome this, we successfully developed a triple-pseudotyped retroviral expression system that can be used for effective gene transfer and expression of reporter genes in both mammalian and penaeid shrimp cells by improving the commercial pantropic retrovirus system (VPK-305, Cell Biolabs) in two ways. First, the promoter regions of translationally controlled tumor protein (TCTP) genes were cloned from the shrimps Metapenaeus ensis and Marsupenaeus japonicus, named Pme-tctp and Pmj-tctp, respectively. We inserted them into the retroviral reporter vectors of pMCs-PLTR-GFP or eGFP, immediately after the retroviral promoter PLTR (5′LTR, long terminal repeat sequence). We then analyzed their expression efficiencies in mammalian and shrimp cells by the lipofection method. It was found that PLTR was a strong promoter in mammalian cells, but failed to effectively drive the reporter genes to express in the primary lymphoid cells (LCOO) derived from Oka organs of shrimp (M. ensis). However, inclusion of the shrimp promoters (Pme-tctp and Pmj-tctp) significantly improved the expression efficiency of the retroviral vectors in shrimp cells. Next, the revised retroviral vectors of pMCs-PLTR-Pme-tctp (or Pmj-tctp)-GFP or eGFP were pseudotyped with envelope protein VSV-G alone, or triply with VSV-G, VP19 and VP28 (two envelope proteins of shrimp white spot syndrome virus). And we then examined the corresponding infection and expression efficiencies in shrimp primary LCOO cells and embryo cells. We found that only the triple-pseudotyped retrovirus containing the shrimp-derived promoter could successfully mediate the delivery and fluorescent expression of GFP and eGFP genes, but VSV-G-pseudotyped retrovirus had poor tropism to shrimp LCOO cells. Confocal microscopic observation further confirmed the hypothesis that the improved tropism of the triple-pseudotyped retrovirus to shrimp cells might be attributed to the inclusion of VP19 and VP28 into the envelope of the packaged retrovirus. In spite of this, the expression efficiency and infection efficiency (20%–30%) of reporter genes in shrimp cells was still lower than those in mammalian cells. In infected shrimp primary embryo cells, strong green fluorescent signal was detected only in neuron-like cells, not in fibroblast-like cells. This triple-pseudotyped retroviral expression system ultimately may prove useful for immortalization of cultured shrimp cells and production of transgenic shrimps. Statement of relevance•We developed a novel triple-pseudotyped retroviral expression system that can be used in effective gene transfer and expression of foreign genes in shrimp cells.•This system may prove useful for the immortalization of cultured shrimp cells which are needed in the study and control of shrimp viral diseases.•This system may prove useful for the production of transgenic shrimps with new traits.

Differential expression of alkaline phosphatase gene in proliferating primary lymphocytes and malignant lymphoid cell lines

Alkaline Phosphatase (APase) activity has been shown to be enhanced specifically in mitogen stimulated B lymphocytes committed to proliferation, but not in T lymphocytes. APase gene expression was analyzed in proliferating murine and human primary lymphocytes and human malignant cell lines using reverse transcriptase and real time PCR. In mitogen stimulated murine splenic lymphocytes, enhancement of APase activity correlated well with an increase in APase gene expression. However, in mitogen stimulated murine T lymphocytes and human PBL despite a vigorous proliferative response, no increase in APase enzyme activity or gene expression was observed. A constitutive expression of APase activity concomitant with APase gene expression was observed inhuman myeloma cell line, U266 B1. However, neither enzyme activity nor gene expression of APase were observed in human T cell lymphoma, SUPT-1. The results suggest a differential expression of APase activity and its gene in proliferating primary lymphocytes of mice and humans. The specific expression of APase activity and its gene only in human myeloma cells, but not in proliferating primary B cells can be exploited as a sensitive disease marker.

Attempts on producing lymphoid cell line from Penaeus monodon by induction with SV40-T and 12S EIA oncogenes.

In an attempt of in vitro transformation, transfection mediated expression of Simian virus-40 (T) antigen (SV40-T) and transduction mediated expression of Adenovirus type 12 early region 1A (12S E1A) oncogene were performed in Penaeus monodon lymphoid cells. pSV3-neo vector encoding SV40-T oncogene and a recombinant baculovirus BacP2-12S E1A-GFP encoding 12S E1A oncogene under the control of hybrid promoters were used. Electroporation and lipofection mediated transformation of SV40-T in lymphoid cells confirmed the transgene expression by phenotypic variation and the expression of GFP in co-transfection experiment. The cells transfected by lipofection (≥ 5%) survived for 14 days with lower toxicity (30%), whilst on electroporation, most of the cells succumbed to death (60%) and survived cells lived up to 7 days. Transduction efficiency in primary lymphoid cells was more than 80% within 14 days of post-transduction, however, an incubation period of 7 days post-transduction was observed without detectable expression of 12S E1A. High level of oncogenic 12S E1A expression were observed after 14 day post-transduction and the proliferating cells survived for more than 90 days with GFP expression, however, without in vitro transformation and immortalization. The study put forth the requirement of transduction mediated 'specific' oncogene expression along with telomerase activation and epigenetic induction for the immortalization and establishment of shrimp cell line.

Emilia sonchifolia extract activity against white spot syndrome virus and yellow head virus in shrimp cell cultures.

Emilia sonchifolia (L.) DC is a plant used in traditional medicine to treat several viral and bacterial diseases. The antiviral activities of selected Sephadex LH-20 column fractions and HPLC subfractions of an acetone extract of E. sonchifolia leaves were determined in shrimp Penaeus merguiensis primary lymphoid cells infected with either white spot syndrome virus (WSSV) or yellow head virus (YHV). WSSV and YHV replication was quantified using quantitative real-time PCR tests targeted to the VP19 and ORF1b gene transcripts, respectively. In lymphoid organ cells exposed to 100 µg ml⁻¹ of either the Sephadex fraction F14 or the HPLC F14 subfraction SF4, both fractions caused reduced replication, but YHV replication was reduced only by SF4. In the asthiazolyl blue mitochondrial enzyme activity assays to assess extract cytotoxicity, >60% of primary lymphoid organ cells remained viable following exposure to 100 µg ml⁻¹ of either F14 or SF4. GC-MS analysis of the HPLC F14 subfraction SF4 showed that it contained 2,4-di-tert-butylphenol. This study is the first to show that E. sonchifolia leaf extracts might be useful as bioactive agents to protect shrimp against viruses such as WSSV and YHV.

Eye extract improves cell migration out of lymphoid organ explants of L. vannamei and viability of the primary cell cultures.

Since no cell line from shrimp has been established up till now, an optimization of the primary cell culture protocol is necessary. In this context, the effect of extracts (supernatant of a 1:50 (w/v) suspension) from different shrimp organs (muscle, brain, ganglia, eyestalk, ovary, and eye) on the performance of primary lymphoid cell cultures was evaluated. Ten percent of eye extract and 3% of ovary extract enhanced maximally the migration and survival of cells of the lymphoid organ of Litopenaeus vannamei significantly at 48, 96, and 144 h post seeding. Extracts from the eyestalk (10%), muscle (10%), and brain (1%) significantly promoted the migration and survival of cells at 48 and 96 h post seeding but not anymore at 144 h post seeding. In conclusion, it may be advised to add 10% of eye extract or 3% of ovary extract to cells for the maximal health of primary cell cultures from the lymphoid organ of L. vannamei.

Problems with the use of liposome- and retrovirus-mediated gene transfer methods in the primary lymphoid cells of the Oka organs of the greasyback shrimp, Metapenaeus ensis (De Haan, 1844)

In this study, both liposome- and retrovirus-mediated gene transfer methods were examined for their potential to transfer and express two retroviral vectors containing the mouse c-Myc or the green fluorescent protein (GFP) gene into the primary lymphoid cell cultures (OKA) derived from “Oka” organs (= organs of the lymphoid system) of the greasyback shrimp Metapenaeus ensis (De Haan, 1844). It was found that the c-Myc gene could be delivered into OKA cells by the liposome-mediated method, but the introduced c-Myc gene could not be effectively transcribed into mRNA. In contrast, the pantropic retrovirus-mediated method failed to introduce the c-Myc gene into OKA cells, and GFP was not detected in the transformed cells, either. This work inferred two problems for the use of the two above-mentioned gene transfer methods in the non-dividing OKA cells: (1) the viral promoter of long terminal repeats (LTRs) had low activity in shrimp cells; (2) the pantropic retrovirus-mediated gene transfer system had a low tropism to shrimp lymphoid cells.

Su1002 Pyrosequencing Analysis of the Synonymous Codon Usage Pattern of rtN236T Mutation in 1418 Cases of Chronic Hepatitis B Patients Treated With Adefovir Dipivoxil

[Background] We previously reported that soluble factors produced from hepatocyte could contribute to the immune pathogenesis of HBV-related inflammation and progression of HCC. (JID2010, JG2012). It has been reported that the progression of fibrosis and HCC is different among the patients infected with various HBV genotypes and HCV. [Aim] The aim of this study is to analyze the effects of chemokines derived from HBV (genotype A, B and C) and HCV -infected hepatocytes and hepatoma cells for the various kinds of immune cells in vitro and in vivo. [Methods] Plasmid Construction: At least 5 clones of each fragment were sequenced and consensus sequence was identified and used as template for 1.24-fold the HBV genome of different genotypes (2 strains each for A, B and C). Cells: Huh7 and HepG2 cells were used for the experiment of transfection. cDNA array: cDNA array for 84chemokine-related genes were carried out. Flow-cytometry analysis: The expression of NKG2D and NKG2A on the NK cells, PD1 on CD8+ cells and MHC class I-related molecule A (MICA) on the HCC cells were analyzed by FACS canto-II. Migration Assay: various kinds of human primary lymphocyte were negatively isolated by MACS beads and used for the migration assay. NOG Mouse model: NOG mice were used for HBV-expressed HCC-transplanted-mouse model. Human primary lymphoid cells were introduced into this mouse. Immunohistochemistry: The liver samples obtained from 20 HBV related HCC patients, HCV-related HCC patients and non HBV/HCV-related HCC patients were stained with CX3CL1, CX3CR1, CD3 and CD56 antibodies. [Results] CCBP2, IL18, CMTM3, CMTM4, CX3CL1, CXCL12, TYMP, GPR81, LTB4R, SDF2 and VHL were well expressed (threshold cycle value range<28) in HBV-replicated-Huh7 cells and significantly up-regulated in comparison to mock transfected Huh7 cells (p<0.01). Among these chemokines-related genes, the expression level of CX3CL1 mRNA was significantly different among genotype A, B and C (relative expression: 1.41, 2.04 and 3.31) (p<0.05). The migration activity of CD16+CD56dimNK cells, CD4+ cells, CD8+ cells and CD14+ Monocyte but not CD19+ B cells or Tregs cells were significantly different among genotype A, B and C in vitro (p<0.05). Moreover, the neutralization of CX3CL1 could cancel these differences of migration activities. Significantly lower expression of NKG2D on CX3CR1+NK cells were observed in primary human peripheral blood mononuclear cells obtained from HBV-related HCC patients and the Tumor-infiltrating lymphocytes in HCC with HBV expression in NOG mouse model in comparison to those is non-HBV related HCC. (p<0.05). [Conclusion] The differential expre s s ion l eve l s o f CX3CL1 and MICA and the d i f f e r en t i a l a t t r ac t i on of NKG2DlowCX3CR1+NK cells and PD-1+ CX3CR1+ CD8 T cells might be involved in HBV persistent infection and hepatocellular carcinoma by affecting immune cells.

Su1000 Hepatoma With HBV Replication Could Attract CX3CR1+ NKG2Dlow NK Cells and CX3CR1+PD-1+ CD8 T Cells

[Background] We previously reported that soluble factors produced from hepatocyte could contribute to the immune pathogenesis of HBV-related inflammation and progression of HCC. (JID2010, JG2012). It has been reported that the progression of fibrosis and HCC is different among the patients infected with various HBV genotypes and HCV. [Aim] The aim of this study is to analyze the effects of chemokines derived from HBV (genotype A, B and C) and HCV -infected hepatocytes and hepatoma cells for the various kinds of immune cells in vitro and in vivo. [Methods] Plasmid Construction: At least 5 clones of each fragment were sequenced and consensus sequence was identified and used as template for 1.24-fold the HBV genome of different genotypes (2 strains each for A, B and C). Cells: Huh7 and HepG2 cells were used for the experiment of transfection. cDNA array: cDNA array for 84chemokine-related genes were carried out. Flow-cytometry analysis: The expression of NKG2D and NKG2A on the NK cells, PD1 on CD8+ cells and MHC class I-related molecule A (MICA) on the HCC cells were analyzed by FACS canto-II. Migration Assay: various kinds of human primary lymphocyte were negatively isolated by MACS beads and used for the migration assay. NOG Mouse model: NOG mice were used for HBV-expressed HCC-transplanted-mouse model. Human primary lymphoid cells were introduced into this mouse. Immunohistochemistry: The liver samples obtained from 20 HBV related HCC patients, HCV-related HCC patients and non HBV/HCV-related HCC patients were stained with CX3CL1, CX3CR1, CD3 and CD56 antibodies. [Results] CCBP2, IL18, CMTM3, CMTM4, CX3CL1, CXCL12, TYMP, GPR81, LTB4R, SDF2 and VHL were well expressed (threshold cycle value range<28) in HBV-replicated-Huh7 cells and significantly up-regulated in comparison to mock transfected Huh7 cells (p<0.01). Among these chemokines-related genes, the expression level of CX3CL1 mRNA was significantly different among genotype A, B and C (relative expression: 1.41, 2.04 and 3.31) (p<0.05). The migration activity of CD16+CD56dimNK cells, CD4+ cells, CD8+ cells and CD14+ Monocyte but not CD19+ B cells or Tregs cells were significantly different among genotype A, B and C in vitro (p<0.05). Moreover, the neutralization of CX3CL1 could cancel these differences of migration activities. Significantly lower expression of NKG2D on CX3CR1+NK cells were observed in primary human peripheral blood mononuclear cells obtained from HBV-related HCC patients and the Tumor-infiltrating lymphocytes in HCC with HBV expression in NOG mouse model in comparison to those is non-HBV related HCC. (p<0.05). [Conclusion] The differential expre s s ion l eve l s o f CX3CL1 and MICA and the d i f f e r en t i a l a t t r ac t i on of NKG2DlowCX3CR1+NK cells and PD-1+ CX3CR1+ CD8 T cells might be involved in HBV persistent infection and hepatocellular carcinoma by affecting immune cells.

Therapeutic Targeting of the Bcl2 Family

Many, perhaps most, cancer chemotherapy agents kill cancer cells via the mitochondrial pathway of apoptosis that is controlled by the Bcl-2 family of proteins. Bcl-2 family proteins regulate commitment to cell death by controlling mitochondrial outer membrane permeabilization (MOMP). To better understand how cancer cells commit to apoptosis, and what drugs might make them commit to apoptosis, we have studied perturbing mitochondria with BH3 peptides that are derived from pro-death Bcl-2 family proteins. Using this provocative test, which we call BH3 profiling, we are able to measure how close a cell is to the threshold of apoptosis, a property we call “priming”. Priming corresponds to sensitivity to chemotherapy. Moreover, BH3 profiling can be used to detect dependence on Bcl-2 and Bcl-xL for survival, which predicts cytotoxic response to small molecule antagonists such as ABT-199 and ABT-263. In acute lymphoblastic leukemia, we find that dependence on either Bcl-2 or Bcl-xL varies from case to case, with very important consequences for sensitivity to ABT-199 and ABT-263. In chronic lymphocytic leukemia, ABT-199 has already demonstrated significant clinical activity that corresponds to its on-target activity in mitochondria in vitro. We have been testing how this in vitro mitochondrial activity in BH3 profiling assays might be translated into a useful clinical predictive biomarker. Finally, we can measure how short term incubation with many kinds of drugs, including targeted pathway inhibitors, can increase cancer cell priming, including for primary lymphoid malignancy cells. This short term increase in priming predicts subsequent cancer cell death, including in clinical treatment. We call this method “Dynamic BH3 Profiling” and are exploring how it might best be utilized in the clinic. Disclosures:Letai:Dana-Farber Cancer Institute: Patents & Royalties; AbbVie: Consultancy.

Detection of Porcine Circovirus Type 2 and Viral Replication by In Situ Hybridization in Primary Lymphoid Organs From Naturally and Experimentally Infected Pigs

Porcine circovirus type 2 (PCV2) infection is the cause of postweaning multisystemic wasting syndrome (PMWS). It has been speculated whether cell types permissive of replication are found in the primary lymphoid organs and whether infection of these tissues has an important role in the pathogenesis of PMWS. The aim of this study was to determine if primary lymphoid organ cells support viral replication during PCV2 infection. This was done by histopathological examination of thymus and bone marrow from pigs experimentally inoculated with PCV2 (n = 24), mock-infected pigs (n = 12), pigs naturally affected by PMWS (n = 33), and age-matched healthy control animals (n = 29). In situ hybridization (ISH) techniques were used to detect PCV2 nucleic acid irrespective of replicative status (complementary probe, CP) or to detect only the replicative form of the virus (replicative form probe, RFP). PCV2 was not detected in the experimentally PCV2-inoculated pigs or the control animals. Among the PMWS-affected pigs, 19 of 20 (95%) thymuses were positive for PCV2 by CP ISH, and 7 of 19 (37%) of these also supported viral replication. By CP ISH, PCV2 was detected in 16 of 33 (48%) bone marrow samples, and 5 of 16 (31%) of these also supported replication. The 2 ISH probes labeled the same cell types, which were histiocytes in both organs and lymphocytes in thymus. The RFP labeled fewer cells than the CP. Thus, PCV2 nucleic acids and replication were found in bone marrow and thymus of PMWS-affected pigs, but there was no evidence that primary lymphoid organ cells are major supporters of PCV2 replication.

The primary culture and subculture of lymphoid cells from shrimp,Penaeus chinensison thermo-sensitive CS/α, β-GP hydrogel

A thermo-sensitive hydrogel composed of chitosan and α, β-glycerophosphate was successfully synthesized and used as basic substrate for primary culture and subculture of lymphoid cells from shrimp, Penaeus chinensis. When the ambient temperature increases from 4°C to 25°C, the chitosan/α, β-glycerophosphate solution transforms into a semi-solid three-dimensional hydrogel with a rough surface. Primary lymphoid cells formed were round and grew homogenously over the surface of the hydrogel. The peroxidase activity indicated that cells seeded on the hydrogel suffered less stress than cells suspended in control culture flasks. The higher relative viability and alkaline phosphatase activity of primary cultured lymphoid cells indicated that the chitosan/α, β-glycerophosphate hydrogel enhanced the growth and proliferation of lymphoid cells. Subsequent subculture of lymphoid cells starting from crumbled primary hydrogel culture was also successful.

Signal Transducer and Activator of Transcription 5b (Stat5b) Serine 193 Is a Novel Cytokine-induced Phospho-regulatory Site That Is Constitutively Activated in Primary Hematopoietic Malignancies

Signal transducer and activator of transcription 5b (Stat5b) is a critical node in the signaling network downstream of external (cytokines or growth factors) or internal (oncogenic tyrosine kinases) stimuli. Maximum transcriptional activation of Stat5b requires both tyrosine and serine phosphorylation. Although the mechanisms governing tyrosine phosphorylation and activation of Stat5b have been extensively studied, the role of serine phosphorylation remains to be fully elucidated. Using mass spectrometry and phospho-specific antibodies, we identified Ser-193 as a novel site of cytokine-induced phosphorylation within human Stat5b. Stat5b Ser(P)-193 was detected in activated primary human peripheral blood mononuclear cells or lymphoid cell lines in response to several γ common (γc) cytokines, including interleukin (IL)-2, IL-7, IL-9, and IL-15. Kinetic and spatial analysis indicated that Stat5b Ser-193 phosphorylation was rapid and transient and occurred in the cytoplasmic compartment of the cell prior to Stat5b translocation to the nucleus. Moreover, inducible Stat5b Ser-193 phosphorylation was sensitive to inhibitors of mammalian target of rapamycin (mTOR), whereas inhibition of protein phosphatase 2A (PP2A) induced phosphorylation of Ser-193. Reconstitution assays in HEK293 cells in conjunction with site-directed mutagenesis, EMSA, and reporter assays indicated that Ser(P)-193 is required for maximal Stat5b transcriptional activity. Indeed, Stat5b Ser-193 was found constitutively phosphorylated in several lymphoid tumor cell lines as well as primary leukemia and lymphoma patient tumor cells. Taken together, IL-2 family cytokines tightly control Stat5b Ser-193 phosphorylation through a rapamycin-sensitive mechanism. Furthermore, constitutive Ser-193 phosphorylation is associated with Stat5b proto-oncogenic activity and therefore may serve as a novel therapeutic target for treating hematopoietic malignancies.

Ex vivo Co-culture of Lymphoid Tissue Stromal Cells and T Cells.

Stromal cells within lymphoid tissues produce IL-7, which is critical for the survival and function of T cells. This protocol is to be used to isolate primary human lymphoid tissue stromal cells to study their impact on the survival of T cells in an ex vivo co-culture system.

Targeting of T/Tn Antigens with a Plant Lectin to Kill Human Leukemia Cells by Photochemotherapy

Photochemotherapy is used both for solid tumors and in extracorporeal treatment of various hematologic disorders. Nevertheless, its development in oncology remains limited, because of the low selectivity of photosensitizers (PS) towards human tumor cells. To enhance PS efficiency, we recently covalently linked a porphyrin (TrMPyP) to a plant lectin (Morniga G), known to recognize with high affinity tumor-associated T and Tn antigens. The conjugation allowed a quick uptake of PS by Tn-positive Jurkat leukemia cells and efficient PS-induced phototoxicity. The present study was performed: (i) to evaluate the targeting potential of the conjugate towards tumor and normal cells and its phototoxicity on various leukemia cells, (ii) to investigate the mechanism of conjugate-mediated cell death. The conjugate: (i) strongly increased (×1000) the PS phototoxicity towards leukemic Jurkat T cells through an O-glycan-dependent process; (ii) specifically purged tumor cells from a 1∶1 mixture of Jurkat leukemia (Tn-positive) and healthy (Tn-negative) lymphocytes, preserving the activation potential of healthy lymphocytes; (iii) was effective against various leukemic cell lines with distinct phenotypes, as well as fresh human primary acute and chronic lymphoid leukemia cells; (iv) induced mostly a caspase-independent cell death, which might be an advantage as tumor cells often resist caspase-dependent cell death. Altogether, the present observations suggest that conjugation with plant lectins can allow targeting of photosensitizers towards aberrant glycosylation of tumor cells, e.g. to purge leukemia cells from blood and to preserve the normal leukocytes in extracorporeal photochemotherapy.