Magnetic-activated Cell Separation Research Articles

Targeted Inhibition of Myeloperoxidase (MPO): A New Therapeutic Strategy for the Treatment of Multiple Myeloma

Despite major improvements in therapeutic strategies for patients with multiple myeloma (MM), effective treatment still remains a persistent challenge, as patients ultimately relapse and succumb to the disease. In the last decade, studies have highlighted the reciprocal interaction between MM plasma cells (PC) and the bone marrow (BM) microenvironment in regulating immune evasion, disease progression and persistence. As MM PC rely on BM stromal cells and their secreted factors for their survival and growth, the therapeutic targeting of the BM microenvironment may prove to be a novel and successful strategy for myeloma care in the future. Myeloid-derived suppressor cells (MDSC), a heterogenous population of myeloid cells are described to promote MM progression through immunosuppression and induction of angiogenesis. Myeloperoxidase (MPO), a key inflammatory enzyme important in host defence, is reported to be the most highly upregulated gene in MDSCs in murine cancer models. Recently, the accumulation of MPO within the tumour microenvironment has attracted much attention with a number of studies describing a role for MPO in regulating cancer development due to its potent pro-oxidative and proinflammatory properties. Our most recent findings have revealed new functional roles for myeloid-derived MPO in the BM microenvironment of MM. Specifically, we demonstrate that myeloid cell populations are increased within the BM of 5TGM1 tumour-bearing mice and that MM PC may directly influence Mpo gene expression in BM-derived myeloid cells. Mechanistically, we report that MPO has the capacity to induce the expression of key MM growth factors, and exerts potent immune suppression by inhibiting anti-tumour T-cell responses. Remarkably, in the syngeneic KaLwRij/5TGM1 mouse model of MM, targeted inhibition of MPO with the suicide substrate 4-Aminobenzoic acid hydrazide (4-ABAH) demonstrated a significant reduction in overall MM tumour burden. Here, we investigate for the first time the efficacy of an orally bioavailable irreversible small molecule inhibitor of MPO (MPOi) in the preclinical Vk*MYC murine model of myeloma. Twelve-week-old C57BL/6J mice were intravenously inoculated with Vk*MYC (Vk14451-GFP) cells and tumour progression was monitored by serum paraprotein electrophoresis (SPEP), whilst endpoint GFP+ tumour cells in the bone marrow were quantitated by flow cytometry. To characterise myeloid cell populations and associated Mpo expression in the Vk*MYC tumour landscape, we utilised flow cytometry to quantitate CD11b+ cells and used magnetic activated cell separation to isolate these populations and characterise the expression of Mpo by qPCR. Our studies confirm that CD11b+ myeloid cells are significantly increased in the BM of Vk*MYC tumour-bearing mice (p<0.01), accompanied by an upregulation of MPO mRNA expression (p<0.001). To assess the efficacy of MPOi in MM, mice were treated with MPOi or vehicle alone twice daily by oral gavage, with treatment initiated at the time of Vk*MYC inoculation. Notably, mice receiving MPOi presented with significantly reduced endpoint tumour burden (9 weeks post tumour cell inoculation). We observed a 15.2% and 31.4% reduction in MM tumour as determined by hind limb GFP% (p<0.05) and SPEP (p<0.01) respectively, compared to vehicle control. Additionally, mRNA analysis of complete BM revealed an upregulation of the critical cytokine IFN gamma (p<0.05) and the downregulation of the potent proangiogenic factor VEGF (p<0.05) in mice treated with MPOi, suggesting MPO inhibition in MM may be an advantageous means to regulate the BM microenvironment and impede disease progression. However, when MPOi treatment was initiated at first signs of detectable disease, (as identified by the presence of a monoclonal spike by SPEP; 5 weeks post tumour cell inoculation), no difference in endpoint tumour burden was observed. This suggests that targeted inhibition of MPO using MPOi may be more effective in at early stages of MM development. In conclusion, the findings presented in this study indicate that inhibiting MPO activity with MPOi, as a single agent therapy, attenuates MM tumour growth in the Vk*Myc mouse model. With limited therapies used in the clinic that target the stromal microenvironment, these findings suggest MPOi could be investigated as a potential novel treatment option that may be included in combination with current frontline therapeutic agents.

Transcriptomic Profiles of Splenic CD19+ B Cells in Mice Chronically Infected With the Larval Echinococcus granulosus.

BackgroundWe previously reported that the larval Echinococcus granulosus (E. granulosus) infection can expand the population of regulatory B cells in mice, thereby inhibiting the anti-infective immunity. However, the underlying mechanism is still largely unknown. This study further investigated the holistic transcriptomic profiles of total splenic B cells following the chronic infection of the parasite.MethodsThe infection model of larval E. granulosus was established by intraperitoneal inoculation with 2000 protoscolexes. Magnetic-Activated Cell Separation (MACS) was used to isolate the total splenic B cells. RNA sequencing was performed to screen the differentially expressed genes (DEGs) after infection. The expression of selected DEGs was verified using qRT-PCR. Gene Ontology (GO) analysis, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis, and Co-expression network analysis were applied to predict these DEGs' underlying biological processes, pathways, and interactions respectively.ResultsA total of 413 DEGs were identified in larval E. granulosus infected B cells, including 303 up- and 110 down-regulated genes. Notably, most DEGs related to inflammation and chemotaxis were significantly upregulated after infection. In line with these changes, significant expression upregulation of DEGs associated with fatty acid oxidation, lipid synthesis, lipolysis, lipid transport, and cholesterol biosynthesis, were observed in infected B cells. Co-expression network analysis showed an intimate interaction between these DEGs associated with immune and metabolism.ConclusionsThe present study revealed that the larval E. granulosus infection induces metabolic reprogramming of B cells, which provides a novel clue to clarify the immunoregulatory mechanism of B cells in parasitic infection.

MACS-DGC versus DGC Sperm Wash Procedure: Comparing Clinical Outcomes in Couples with Male Factor Infertility Undergoing ICSI: A Clinical Trial Study.

BackgroundImplementation of sperm preparation techniques based on cellular and molecular characteristics can improve the clinical outcomes of couples with male factor infertility. These methods attempt to select better sperm compared to classical methods of preparation such as swim-up and density gradient centrifugation (DGC). In this view, the aim of this study was the comparison of clinical outcomes of magnetic-activated cell separation (MACS) followed by DGC or DGC alone in infertile men undergoing intracytoplasmic sperm injection (ICSI).Materials and MethodsFor this prospective single parallel blind clinical trial study, 206 infertile couples with male factor infertility and having abnormal sperm morphology higher than 96% were included. 106 and 100 couples were considered for the study (MACS-DGC) and control group (DGC), respectively. Clinical outcomes of ICSI; fertilization, embryo quality, and implantation, pregnancy rates were compared between two groups.Materials and MethodsFor this prospective single parallel blind clinical trial study, 206 infertile couples with male factor infertility and having abnormal sperm morphology higher than 96% were included. 106 and 100 couples were considered for the study (MACS-DGC) and control group (DGC), respectively. Clinical outcomes of ICSI; fertilization, embryo quality, and implantation, pregnancy rates were compared between two groups.ResultsMean of fertilization (80.19 ± 1.88 vs. 75.63 ± 2.06, P=0.1), top embryo quality on the day 3 (30.22 ± 3.59 vs. 17.96 ± 2.9, P=0.009), clinical pregnancy (30.76% vs. 22.22%, P=0.19), and implantation rate (18.12% vs. 10.42%, P=0.04) were higher in the study group compared to the control group.ConclusionSperm preparation by MACS followed by DGC in teratozoospermic men could improve the clinical outcomes after ICSI (Registration number: IRCT201610317223N8).

CDK9 Inhibition with LDC526 Is an Effective Treatment for T-Cell Prolymphocytic Leukemia (T-PLL) in Vitro

▪IntroductionT-cell prolymphocytic leukemia (T-PLL) is a rare leukemia with an aggressive disease course, characterized by chemotherapy resistance and a median overall survival of less than two years. Apart from defects in DNA damage response genes like ATM and genes of JAK/STAT signaling, overexpression of MYC, often caused by gain of chromosomal material, is frequently observed in T-PLL. Intravenous application of the anti-CD52 antibody Alemtuzumab has been demonstrated to improve progression-free survival and is currently considered the standard of care. In younger patients remissions can be consolidated by allogeneic stem cell transplantation, which results in long-term remission in a small subgroup of patients. The prognosis in the majority of patients relapsing from their disease is dismal and thus novel treatment options are urgently needed. Cyclin-dependent kinase 9 (CDK9), a serine/ threonine kinase which is ubiquitously expressed, is the catalytic unit of the positive transcription elongation factor (P-TEFb). In the presence of cyclin T, p-TEFb stimulates transcription elongation by phosphorylation of the carboxy-terminal domain (CTD) of RNA polymerase II (RNA Pol II). CDK9 inhibition by LDC526, a specific CDK9 inhibitor, leads to reduced phosphorylation of CTD-Ser2, Ser5 and Ser7 of RNA Pol II and subsequently to the downregulation of MYC and MCL-1.Methods</spanCD3+CD4+ tumor cells from cryopreserved peripheral blood samples of 11 T-PLL patients were enriched to a purity of >95% by fluorescence-activated cell sorting (FACS) or magnetic activated cell separation (MACS). RNA and DNA were isolated using the RNeasy Mini Kit® or the QIAamp DNA blood midi kit® (Qiagen, Hilden, Germany), respectively. To determine IC50 values, we performed cell viability assays for T-PLL cells of 11 patients treated with LDC526 and a panel of reference compounds compared to DMSO controls (CellTiter-Glo Luminescent Cell Viability Assay; Promega, Fitchburg, WI, USA). Gene expression profiling was performed in primary T-PLL cells treated with the CDK9 inhibitor LDC526 in a concentration of 10µM compared to DMSO (n=3) on Clariom S Pico Assays (Affymetrix, Santa Clara, CA, USA) and analyzed by gene set enrichment analysis (GSEA) and GeneTrail analysis. Gene expression measurement was performed with quantitative real-time PCR (qPCR) for MYC (Taqman, Thermo Fisher Scientific, Waltham, MA, USA). Protein expression was analyzed for pCTD Ser2, Ser5 and Ser7, MYC, p-STAT3 and MCL-1 by Western Blot (n=4) after LDC526-treatment in three different concentrations (10, 30 and 100 µM).ResultsLDC526 inhibited T-PLL cell survival in vitro in a dose-dependent manner. The median IC50 was 1.7 µM (range: 0.9 -3.5) which compared favourably to a panel of reference compounds including fludarabine, clofarabine, methotrexate, cytarabine and ruxolitinib (median IC50 values 6.4, 6.2, 29.9, 8.3, and 29.2, respectively). Similar experiments with BAY1143572, a structurally closely related CDK9 inhibitor, which is currently investigated in phase I studies of acute myeloid leukemia, revealed an even lower median IC50 of 0.9 µM (range: 0.5 - 1.7). Of note, the purine analogues fludarabine and clofarabine were only active in some of the patients tested, whereas both CDK9 inhibitors showed activity in all samples analyzed.Gene expression profiles of LDC526 treated T-PLL cells compared to DMSO controls revealed an enrichment of genes in gene sets associated with interferon and TNFA signatures, MYC targets as well as JAK/STAT genes, which were subsequently confirmed by qPCR experiments. At the protein level LDC526 dose-dependently reduced phosphorylation of CTD-Ser2, Ser7 and to a lesser extent of Ser5 compared to DMSO controls resulting in efficient down-regulation of MYC, MCL-1 and p-STAT3.ConclusionsOur experiments identify CDK9 as a novel therapeutic target in T-PLL which can be efficiently inhibited by LDC526 and BAY1143572 at clinically meaningful concentrations. CDK9 inhibition resulted in both reduced T-PLL cell survival and down-regulation of MYC and JAK/STAT signaling, which have a key role in the molecular pathogenesis of T-PLL. DisclosuresDürig:Lead Discovery Center: Research Funding.

Dielectrophoretic high-purity isolation of primary T-cells in samples contaminated with leukemia cells, for biomanufacturing of therapeutic CAR T-cells

Chimeric antigen receptor (CAR) T-cell therapy is a Food and Drug Administration (FDA)-approved treatment for specific cancers. CAR T-cells are manufactured ex vivo using T-cells derived from patients. Recent clinical trials reported a very high level of efficacy as well as complete remission rates (70%–93%) for acute lymphoblastic leukemia (ALL) patients. However, there have also been reports of significant adverse effects resulting from the contamination of T-cells derived from patients with existing cancer cells during manufacturing. The accidental transfection of cancer cells with CAR genes provides the cancer cells with immunity from CAR T-cells. As a result, genetically modified cancer cells that express the CAR molecules grow into secondary tumors. This significantly undermines the great benefits offered by CAR T-cell therapy. The removal of cancer cells before gene transfer using cell purification methods can address this issue. However, traditional cell purification methods such as fluorescence-activated cell separation and magnetic-activated cell separation have proven to be unsuitable for high-purity T-cell purification. To address this issue, we investigated the utility, in microfluidic channels, of dielectrophoretic cell purification, which is a label-free high-throughput cell purification method. When we conducted the experiments with primary T-cell (CD8+) samples contaminated with cultured acute lymphoblastic leukemia and chronic myelogenous leukemia cells, we found that dielectrophoresis could be used to purify cell samples and achieve 100% purity with high cell viability (greater than 90%).

Preclinical and Clinical Evaluation of Magnetic-Activated Cell Separation Technology for CTC Isolation in Breast Cancer.

Circulating tumor cell (CTC) count is an independent prognostic factor in early breast cancer. CTCs can be found in the blood of 20% of patients prior to neoadjuvant therapy. We aimed to assess the suitability of magnetic-activated cell separation (MACS) technology for isolation and cytological characterization of CTCs. In the preclinical part of the study, cell lines were spiked into buffy coat samples derived from healthy donors, and isolated using MACS. Breast cancer cells with preserved cell morphology were successfully isolated. In the clinical part, blood for CTC isolation was drawn from 44 patients with early and locally advanced breast cancer prior to neoadjuvant chemotherapy. Standard Giemsa, Papanicolaou and pancytokeratin staining was applied. 2.3% of samples contained cells that meet both the morphological and immunocytochemical criteria for CTC. In 32.6% of samples, partially degenerated pancytokeratin negative cells with morphological features of tumor cells were observed. In 65.1% of samples, CTCs were not found. In conclusion, our results demonstrate that morphologically intact tumor cells can be isolated using MACS technology. However, morphologically intact tumor cells were not detected in the clinical part of the study. At present, MACS technology does not appear suitable for use in a clinical cytopathology laboratory.

Decrease of spermatozoa with an unbalanced chromosome content after cell sorting in men carrying a structural chromosomal abnormality.

We showed that in men with a constitutional chromosomal abnormality, DNA fragmentation was significantly higher in chromosomally unbalanced spermatozoa than in spermatozoa with a normal or balanced chromosomal content. These results could be explained by a phenomenon already described in infertile men: abortive apoptosis. To determine whether magnetic-activated cell separation could select spermatozoa with lower levels of DNA fragmentation and unbalanced chromosome content in men carrying a structural chromosomal abnormality. The spermatozoa of ten males with a chromosomal rearrangement were separated into two populations using magnetic-activated cell separation (annexin V (-) and annexin V (+) fractions), in order to study meiotic segregation by fluorescence insitu hybridization, the percentage of spermatozoa with an externalization of phosphatidylserine by annexin V staining and DNA fragmentation by TdT-mediated dUTP nick-end labeling on the whole ejaculate and on selected spermatozoa in the same patient. For all patients, the percentage of spermatozoa with externalization of phosphatidylserine decreased in the annexin V (-) fraction and increased in the annexin V (+) fraction as compared to the frozen-thawed semen sample. The rates of DNA fragmentation were statistically much lower in the annexin V (-) fraction when compared to the rate before magnetic-activated cell separation for all but one patient. Conversely, we observed a statistically significantly higher rate of DNA fragmentation in the annexin V (+) fraction for six patients. After magnetic-activated cell separation, there was a significant increase of normal/balanced spermatozoa in the fraction of annexin V (-) for all patients. Conversely, we observed a significant decrease in the fraction of annexin V (+) for seven patients. Magnetic-activated cell separation is a promising tool for increasing the selection of healthy spermatozoa, with a decrease in the number of spermatozoa with externalization of phosphatidylserine, DNA fragmentation, and chromosome unbalance, for use in assisted reproductive technologies such as intracytoplasmic sperm injection for males with a chromosomal structural abnormality.

Expression of Tmem119/Sall1 and Ccr2/CD69 in FACS-Sorted Microglia- and Monocyte/Macrophage-Enriched Cell Populations After Intracerebral Hemorrhage.

Activation and polarization of microglia and macrophages are critical events in neuroinflammation and hematoma resolution after intracerebral hemorrhage (ICH). However, distinguishing microglia and monocyte-derived macrophages histologically can be difficult. Although they share most cell surface markers, evidence indicates that the gene regulation and function of these two cell types might be different. Flow cytometry is the gold standard for discriminating between the two cell populations, but it is rarely used in the ICH research field. We developed a flow cytometry protocol to identify and sort microglia and monocyte-derived macrophages from mice that have undergone well-established ICH models induced by collagenase or blood injection. In addition, we combined a recently established magnetic-activated cell separation system that allows eight tissue samples to be assessed together. This protocol can be completed within 5–8 h. Sorted cells are fully preserved and maintain expression of microglia-specific (Tmem119/Sall1) and macrophage-specific (Ccr2/CD69) markers. They retain phagocytic ability, respond to lipopolysaccharide stimulation, and engulf fluorescent latex beads. Thus, this protocol represents a very important tool for researching microglial and monocyte-derived macrophage biologic function after ICH and other brain diseases.

Enrichment of Adipose-Derived Stromal Cells for BMPR1A Facilitates Enhanced Adipogenesis.

Reconstruction of soft tissue defects has traditionally relied on the use of grafts and flaps, which may be associated with variable resorption and/or significant donor site morbidity. Cell-based strategies employing adipose-derived stromal cells (ASCs), found within the stromal vascular fraction (SVF) of adipose tissue, may offer an alternative strategy for soft tissue reconstruction. In this study, we investigated the potential of a bone morphogenetic protein receptor type 1A (BMPR1A)(+) subpopulation of ASCs to enhance de novo adipogenesis. Human lipoaspirate was enzymatically digested to isolate SVF and magnetic-activated cell separation was utilized to obtain BMPR1A(+) and BMPR1A(-) cells. These cells, along with unenriched cells, were expanded in culture and evaluated for adipogenic gene expression and in vitro adipocyte formation. Cells from each group were also labeled with a green fluorescent protein (GFP) lentivirus and transplanted into the inguinal fat pads, an adipogenic niche, of immunocompromised mice to determine their potential for de novo adipogenesis. Confocal microscopy along with staining of lipid droplets and vasculature was performed to evaluate the formation of mature adipocytes by transplanted cells. In comparison to BMPR1A(-) and unenriched ASCs, BMPR1A(+) cells demonstrated significantly enhanced adipogenesis when cultured in an adipogenic differentiation medium, as evidenced by increased staining with Oil Red O and increased expression of peroxisome proliferator-activating receptor gamma (PPAR-γ) and fatty acid-binding protein 4 (FABP4). BMPR1A(+) cells also formed significantly more adipocytes in vivo, as demonstrated by quantification of GFP+ adipocytes. Minimal formation of mature adipocytes was appreciated by BMPR1A(-) cells. BMPR1A(+) ASCs show an enhanced ability for adipogenesis in vitro, as shown by gene expression and histological staining. Furthermore, within an adipogenic niche, BMPR1A(+) cells possessed an increased capacity to generate de novo fat compared to BMPR1A(-) and unenriched cells. This suggests utility for the BMPR1A(+) subpopulation in cell-based strategies for soft tissue reconstruction.

Altered Suppressor Function of Regulatory T Cells in Type 1 Diabetes.

Type 1 diabetes (T1D) is a T cell mediated autoimmune disease targeting the insulin-producing β cells within pancreatic islets. Autoimmune diseases may develop as a consequence of altered balance between regulatory (Tregs) and autoreactive T cells. To evaluate Treg cells frequency and suppressive function in the peripheral blood of newly diagnosed T1D patients in comparison with healthy controls. Fifteen new cases of T1D patients and 15 age- and sex- matched healthy controls were recruited to this study. Their peripheral blood mononuclear cells (PBMCs) were isolated and CD4+CD25+FoxP3+CD127-/low Treg cells were studied by flowcytometry technique. Thereafter, Tregs were isolated by Magnetic-Activated Cell Separation (MACS) technology and by using CFSE (carboxyfluorescein succinimidyl ester) dilution assay, their suppressive activity was evaluated in the coculture of CD4+CD25- T responder cells with Treg cells. The percentage of CD4+CD25+FoxP3+CD127-/low Tregs did not differ between T1D patients and healthy controls but the MFI (mean fluorescence intensity) of transcription factor FoxP3 (forkhead box protein P3) was significantly decreased in T1D patients (20.03 ± 1.4 vs. 31.33 ± 2.95, p=0.0017). Moreover, the suppressive function of CD4+CD25+CD127-/low Treg cells was significantly diminished in T1D patients in comparison with control group (35.16 ± 4.93% vs. 60.45 ± 5.26%, respectively, p=0.0015). Present study indicates an impaired immune regulation among T1D patients, characterized by defects in suppressive function and expression of FoxP3 in Treg cells without any significant decrease in their frequency in peripheral blood.

Isolation of mast cells from the peritoneal exudate of the teleost fish gilthead sea bream (Sparus aurata L.)

Inflammation is the first response of animals to infection or tissue damage. Sparus aurata (Perciformes) was the first fish species shown to possess histamine-containing mast cells at mucosal tissues. We report a separation protocol for obtaining highly enriched (over 95% purity) preparations of fish mast cells in high numbers (5-20 million mast cells per fish). The peritoneal exudate of S. aurata is composed of lymphocytes, acidophilic granulocytes, macrophages and mast cells. We separated the lymphocyte fraction through discontinuous density gradient centrifugation. The remaining cells were cultivated overnight in RPMI-1640 culture medium containing 5% fetal calf serum, which allowed macrophages to adhere to the cell culture flasks. Finally, acidophilic granulocytes were separated from the mast cells though a Magnetic-Activated Cell Separation (MACS) protocol, using a monoclonal antibody against these cells. The purity of mast cells-enriched fractions was analyzed by flow cytometry and by transmission electron microscopy. The functionality of purified mast cells was confirmed by the detection of histamine release by ELISA after stimulation with compound 48/80 and the induction of the pro-inflammatory cytokines IL-1β and IL-8 following stimulation with bacterial DNA. This fish mast cells separation protocol is a stepping stone for further studies addressing the evolution of vertebrate inflammatory mechanisms.

Toll-Like Receptors 7, 8, and 9 Expression and Function in Primary Human Cervical Cancer Langerhans Cells: Evidence of Anergy

Human papillomavirus oncoproteins E6 and E7 down modulate Toll-like receptor (TLR) 9 expression in infected keratinocytes. We explored the status of expression and function of TLR7, TLR8, and TLR9 in primary human Langerhans cells (LCs) isolated from cervical tumors. Single-cell suspensions were made from fresh tissues of squamous cell carcinoma (International Federation of Gynecology and Obstetrics stage IB2); myeloid dendritic cells were purified using CD1c magnetic activated cell separation kits. Langerhans cells were further flow sorted into CD1a*CD207* cells. Acute monocytic leukemia cell line THP-1-derived LCs (moLCs) formed the controls. mRNA from flow-sorted LCs was reverse transcribed to cDNA and TLR7, TLR8, and TLR9 amplified. Monocyte-derived Langerhans cells and cervical tumor LCs were stimulated with TLR7, TLR8, and TLR9 ligands. Culture supernatants were assayed for interleukin (IL) 1β, IL-6, IL-10, IL-12p70, interferon (IFN) α, interferon γ, and tumor necrosis factor (TNF) α by Luminex multiplex bead array. Human papillomavirus was genotyped. We have for the first time demonstrated that the acute monocytic leukemia cell line THP-1 can be differentiated into LCs in vitro. Although these moLCs expressed all the 3 TLRs, tumor LCs expressed TLR7 and TLR8, but uniformly lacked TLR9. Also, moLCs secreted IL-6, IL-1β, and tumor necrosis factor α to TLR8 ligand and interferon α in response to TLR9 ligand; in contrast, tumor LCs did not express any cytokine to any of the 3 TLR ligands. Human papillomavirus type 16 was one of the common human papillomavirus types in all cases. Cervical tumor LCs lacked TLR9 expression and were functionally anergic to all the 3: TLR7, TLR8, and TLR9 ligands, which may play a crucial role in immune tolerance. The exact location of block(s) in TLR7 and TLR8 signaling needs to be investigated, which would have important immunotherapeutic implications.

ATL

Analysis of tumor-infiltrating lymphocytes (TILs) is one of the cornerstones for the understanding of immune responses prevailing in the tumor microenvironment. We studied TILs from squamous cell carcinoma of the cervix ex vivo without proliferating them in vitro before analysis. Whereas TILs were magnetic activated cell separation enriched and flow sorted into CD4 CD25 (regulatory T cells [Tregs]), CD4 CD25 (effector T cells [Teffs]) were directly purified by flow cytometry, and both these subsets were characterized phenotypically and functionally. Tissue sections were probed for interleukin 4 (IL-4) and interferon γ. Effector T cells constitutively expressed both interferon γ and IL-4 prototypical cytokines of TH1 and TH2, respectively, and were able to proliferate and secrete higher quantities of both cytokines in response to anti-CD3/anti-CD28 and autologous tumor lysates. Only 53% of cervical cancer Tregs were FOXP3, elaborated transforming growth factor β1, and IL-10 and were able to inhibit both T helper subsets. Intratumoral Teffs represented functionally active subsets of both TH1 and TH2 that were not anergic but were suppressed by multiple Treg subsets, which comprised FOXP3 + Tregs and Tregs secreting transforming growth factor β1 and IL-10. These results imply that the microenvironment of cervical carcinomas harbored both TH1 and TH2 subsets of CD4 Teffs that were functionally active but were perhaps unable to perform because of the overpowering effect of Tregs.

Circulating melanoma cells isolated from clinical blood samples and characterized by full-length mRNA sequencing at single-cell level.

10539 Background: Melanoma is the most aggressive type of skin cancer. Late-stage melanoma is highly metastatic and currently lacks effective treatment. This discouraging clinical observation highlights the need for a better understanding of the molecular mechanisms underlying melanoma initiation and progression and for developing new therapeutic approaches based on novel targets. Although genome-wide transcriptome analyses have been frequently used to study molecular alterations in clinical samples, it has been technically challenging to obtain the transcriptomic profiles at single-cell level. Methods: Using antibody-mediated magnetic activated cell separation (MACS), we isolated and individualized putative circulating melanoma cells (CMCs) from the blood samples of the melanoma patients at advance stages. The transcriptomic analysis based on a novel and robust mRNA-Seq protocol (Smart-Seq) was established and applied to the putative CMCs for single-cell profiling. Results: We have discovered distinct gene expression patterns, including new putative markers for CMCs. Meanwhile, the gene expression profiles derived of the CMC candidates isolated from the patient’s blood samples are closely-related to the expression profiles of other cells originated from human melanocytes, including normal melanocytes in primary culture and melanoma cell lines. Compared with existing methods, Smart-Seq has improved read coverage across transcripts, which provides advantage for better analyzing transcript isoforms and SNPs. Conclusions: Our results suggest that the techniques developed in this research for cell isolation and transcriptomic analyses can potentially be used for addressing many biological and clinical questions requiring genomewide transcriptome profiling in rare cells.

Identity, Potency,In VivoViability, and Scaling Up Production of Lentiviral Vector-Induced Dendritic Cells for Melanoma Immunotherapy

SmartDCs (Self-differentiated Myeloid-derived Antigen-presenting-cells Reactive against Tumors) consist of highly viable dendritic cells (DCs) induced to differentiate with lentiviral vectors (LVs) after an overnight ex vivo transduction. Tricistronic vectors co-expressing cytokines (granulocyte-macrophage-colony stimulating factor [GM-CSF], interleukin [IL]-4) and a melanoma antigen (tyrosine related protein 2 [TRP2]) were used to transduce mouse bone marrow cells or human monocytes. Sixteen hours after transduction, the cells were dispensed in aliquots and cryopreserved for identity, potency, and safety analyses. Thawed SmartDCs readily differentiated into highly viable cells with a DC immunophenotype. Prime/boost subcutaneous administration of 1×10(6) thawed murine SmartDCs into C57BL/6 mice resulted into TRP2-specific CD8(+) T-cell responses and protection against lethal melanoma challenge. Human SmartDC-TRP2 generated with monocytes obtained from melanoma patients secreted endogenous cytokines associated with DC activation and stimulated TRP2-specific autologous T-cell expansion in vitro. Thawed human SmartDCs injected subcutaneously in NOD.Rag1(-/-).IL2rγ(-/-) mice maintained DC characteristics and viability for 1 month in vivo and did not cause any signs of pathology. For development of good manufacturing practices, CD14(+) monocytes selected by magnetic-activated cell separation were transduced in a closed bag system (multiplicity of infection of 5), washed, and cryopreserved. Fifty percent of the monocytes used for transduction were recovered for cryopreservation. Thawed SmartDCs produced in two independent runs expressed the endogenous cytokines GM-CSF and IL-4, and the resulting homogeneous SmartDCs that self-differentiated in vitro contained approximately 1.5-3.0 copies of integrated LVs per cell. Thus, this method facilitates logistics, standardization, and high recovery for the generation of viable genetically reprogrammed DCs for clinical applications.

Immunocytochemical characterisation of cultures of human bladder mucosal cells

BackgroundThe functional role of the bladder urothelium has been the focus of much recent research. The bladder mucosa contains two significant cell types: urothelial cells that line the bladder lumen and suburothelial interstitial cells or myofibroblasts. The aims of this study were to culture these cell populations from human bladder biopsies and to perform immunocytochemical characterisation.MethodsPrimary cell cultures were established from human bladder biopsies (n = 10). Individual populations of urothelial and myofibroblast-like cells were isolated using magnetic activated cell separation (MACS). Cells were slow growing, needing 3 to 5 weeks to attain confluence.ResultsCytokeratin 20 positive cells (umbrella cells) were isolated at primary culture and also from patients' bladder washings but these did not proliferate. In primary culture, proliferating cells demonstrated positive immunocytochemical staining to cytokeratin markers (AE1/AE3 and A0575) as well fibroblasts (5B5) and smooth muscle (αSMA) markers. An unexpected finding was that populations of presumptive urothelial and myofibroblast-like cells, isolated using the MACS beads, stained for similar markers. In contrast, staining for cytokeratins and fibroblast or smooth muscle markers was not co-localised in full thickness bladder sections.ConclusionsOur results suggest that, in culture, bladder mucosal cells may undergo differentiation into a myoepithelial cell phenotype indicating that urothelial cells have the capacity to respond to environmental changes. This may be important pathologically but also suggests that studies of the physiological function of these cells in culture may not give a reliable indicator of human physiology.

Differential Genomic and Proteomic Signatures In Cord Blood (CB) Vs Peripheral Blood (PB) CD56+Dim NK Cells: Over Expression of CD34 In CB Vs PB CD56+Dim NK Cells

AbstractAbstract 2781 Background.Umbilical cord blood (UCB) is a viable alternative source of allogeneic hematopoietic stem cells for the treatment of both malignant and non-malignant disease (Cairo et al BBMT 2008). UCB transplantation (UCBT) is known to be associated with decrease severe acute graft-versus-host disease (GvHD) compared to unrelated bone marrow (BM) and peripheral blood (PB) transplantation; however, it is associated with delayed hematopoietic and immune reconstitution (Szabolcs/Cairo et al Seminars in Hematology 2010). NK cells play important roles in both innate and adaptive immunity and are characterized as a CD56+ cell population. NK cell recovery is prompt by 2 months after hematopoietic stem cell transplantation (HSCT), while T-cell (after at least 9 mo HSCT) and B-cell (after 3 to 4 mo HSCT) reconstitutions are gradual and delayed. CD56+dim cells are primarily cytotoxic and make up 90% of PB NK populations (Shereck/Cairo PBC 2007). We previously demonstrated the ability to ex-vivo expand CB MNC into various phenotypes of CD56+dim and CD56+bright NK cells (totally 60%) and NKT cells (40%) with profound in vitro and in vivo cytotoxicity against hematological malignancies (Ayello/Cairo BBMT 2006 & Exp. Hematology 2009). Proteomic studies from our group demonstrated differential protein expression including ↑NKG2A, ↓IP3R type 3, ↓MAPKAPK5, and ↑NOTCH 2 in CB vs PB CD56+dim (Shereck/Cairo, ASH 2007; Shereck/Day/Cairo, ASBMT 2009). Objective.In these studies, we sought to determine the similarity or differences in genetic signatures in CB vs APB CD56+dim NK cells. Methods.CB MNCs were isolated on a ficoll gradient and NK CD56+16+dim cells isolated using a 2-step magnetic activated cell separation (MACS) process via a standard kit (Miltenyi Biotec). Enrichment was at least 94%. Isolated RNA from CB and PB CD56+dim cells were subjected to microarray studies (Affymetrix, U133A_2) as we have previously described (Jiang/Cairo et al J Immunol 2004). Data were analyzed by Agilent GeneSpring and Ingenuity pathway analyses. Welch test were used to perform statistical analysis and fold change of < 1.5 and values of p<0.05 were considered to be significant. Two-color ECL Plex fluorescence Western blotting (WB) was preformed to validate the proteomic data. Protein samples were separated using SDS-PAGE followed by transblotting. WB membranes were then incubated with target and control (GAPDH) primary antibodies. After rinse and wash, the membranes were further incubated with CY5 and CY3 conjugated secondary antibodies. The membranes were scanned with TYPHOON by green (532 laser and 580 filter) and red (633 laser and 670 filter) setting for CY3 and CY5, respectively, and then observed and quantified using ImageQuant. Results.CB vs PB CD56+dim cells significantly altered expressed 796 genes, in which 486 genes were over expressed, at the genomic level including: pro-apoptotic genes: CASP10 (3.1F), TNFSF11 (4.7F), CDC2 (3.0F), BCL2L1 (4.3F), NOTCH2 (1.5F); and cell development: PBX1 (7.6F), IL1RN (5.1F), CD24 (5.3F), CD34 (3.5F), CD55 (2.1F), CCL13 (2.2F). Conversely, there was significant under expression of NF1 (5.1F), MAP2K3 (1.7F), PIK3CD (2.1F), BAX (2.9F), and JUN (2.2F). Our WB results indicate that NOTCH2 (2.4F) and PBX1 (2.2F) proteins are increased in CB vs PB CD56+dim NK cells, consistent with our proteomic results. Conclusion.These results suggest that CB vs PB CD56+dim NK are more prone to undergo programmed cell death (apoptosis) secondary to over expression of numerous pro-apoptotic genes, and may be earlier in development (pro-NK) with over expression of the CD34 gene. Furthermore, decrease CB vs PB NK cytotoxicity maybe in part secondary to increase programmed cell death in particularly increase NOTCH2 at the genomic and proteomic levels. (The first two authors contribute equally.) Disclosures:No relevant conflicts of interest to declare.

Occurrence of Chromosomal Abnormalities In Bone Marrow CD14+ monocytes of Multiple Myeloma Patients

AbstractAbstract 2968 Introduction:Increased bone marrow angiogenesis is a characteristic feature of multiple myeloma (MM). New blood vessels formation is provided by recruiting vascular endothelial cells from existing capillaries or circulating endothelial progenitor cells (EPC). EPCs have distinct monocytic features and can be cultured from CD14+ cells. In addition, monocytes were shown to contribute to angiogenesis as EPCs in vivo. It has been established that myeloid progenitor cells participate in the development of endothelial precursors. In MM, it has been reported that circulating EPCs carried the same chromosomal aberrations as neoplastic plasma cells (PC). It is possible that EPCs originate from common precursor that gives rise to both PCs and endothelial cells. The purpose of our pilot study was to clarify whether myeloid cells, CD14+ monocytes (CD14+MO), in MM might carry the same chromosomal abnormalities as CD138+PC. Methods:Total of 15 MM patients were enrolled in this pilot study; 93 % (14/15) typical MM; 7 % (1/15) plasma cell leukemia (PCL). CD138+PCs were isolated by magnetic-activated cell separation (MACS) from bone marrow mononuclear cells (BMMNC). CD14+MOs were isolated by fluorescence-activated cell sorting (FACS) from BMMNC after isolation of CD138+PCs. The purity of both cell populations was > 90%. Distinct BM cell fractions were analyzed for del(13q14), del(17p53), IgH gene rearrangement, hyperdiploidy and trisomy of chromosomes 5, 9, 15 by interphase fluorescence in situ hybridization (FISH) according to standard protocol. The cut-off level for detection of choromosomal aberarations was set to 20%. Results:FISH analysis proved at least one of studied chromosomal abnormalities in 20% (3/15) of MM patients (See Table 1; No.1 – No.3). In case No.1, a patient with PCL, CD14+MOs were positive for del(13q14)(31%), IgH gene rearrangement (46%) and trisomy 9 (47%). The same aberrations were found in CD138+PCs but with higher frequency (Table 1). Patient No.2 was positive for del(13q14)(35%) and for del(17p53)(39%) in CD14+MO but in CD138+PCs, the patient was positive further for hyperdiploidy (53%) and trisomy 5 (28%), 9 (56%) and 15 (60%). CD14+MOs of patient No.3 were positive only for trisomy 15 (25%) which corresponded to trisomy 15 (24%) in CD138+PCs. Other chromosomal abnormalities found in CD138+PCs of No.3 were not detected in CD14+MOs. Chromosomal aberrations identified in CD14+MOs of No.4 – No.15 did not reach the cut-off level for positivity (Table 1). Conclusion:In our pilot study, we demonstrated for the first time that bone marrow CD14+ monocytes of MM patients carried the same chromosomal aberrations as CD138+ plasma cells. The possible explanation is that CD14+MOs and CD138+PCs may origin from a common precursor. We hypothesize that myeloid cells might play a role in blood vessel formation as endothelial precursors and might be important in pathogenesis of multiple myeloma. It requires further study to elucidate the relationship between two distinct bone marrow populations and a potential role of CD14+cells in disease severity. Supported with research program MSM of Czech republic Nr. 0021622434 and LC06027. Disclosures:No relevant conflicts of interest to declare.

Antibody-Dependent Enhancement of Coxsackievirus B3 Infection of Primary CD19+B Lymphocytes

Coxsackievirus B3 (CVB3) is associated with several different acute and chronic forms of human disease, including myocarditis, aseptic meningitis, and pancreatitis. Moreover, CVB3 also infects immune cells like CD19+ B lymphocytes, but the viral uptake mechanism into these cells is not well understood. Therefore, primary murine and human CD19+ B cells were isolated by magnetic-activated cell separation technology and analyzed for virus receptor expression, antibody-dependent enhancement of viral infection, and different cellular surface proteins, that might be involved in mechanisms of viral uptake. Western blot analysis of these cells revealed no significant expression of the coxsackievirus-adenovirus receptor CAR. But incubation of CVB3 with serum dilutions, which exhibited binding but not neutralizing characteristics, increased viral uptake and replication significantly in a dose-dependent manner. Viral entry was reduced when Fc portions of immunoglobulins were blocked by protein A treatment. Moreover, the classical complement system rather than Fc-gamma-receptor-mediated mechanisms could be involved in viral uptake. Taken together, these data suggest an antibody-dependent enhancement of CVB3 infection of primary murine and human CD19+ B cells.

Long‐term proliferation and characterization of human spermatogonial stem cells obtained from obstructive and non‐obstructive azoospermia under exogenous feeder‐free culture conditions

The aim of the present study was to improve efficiency of isolation and to optimize proliferative potential of human spermatogonial stem cells (SSCs) obtained from obstructive azoospermic (OA) and non-obstructive azoospermic (NOA) patients, and further, to characterize these cells for potential use in infertility treatment or study of reproductive biology. We have applied a cell-sorting method, using collagen and magnetic activated cell separation to overcome obstacles, developing a collection system, and simple long-term proliferation system, that yields large numbers of high-purity SSCs from obstructive OA and NOA patients. SSCs derived from OA and NOA patients proliferated and maintained their characteristics for more than 12 passages (>6 months) in vitro. Moreover, the population of cells positive for the SSC-specific markers GFRalpha-1 and integrin alpha6, increased to more than 80% at passage 8. These finding may support the idea that in vitro propagation of SSCs could be a useful tool for infertility treatment and study of reproductive biology.