Adherent Peripheral Blood Mononuclear Cells Research Articles

Osteostatin Inhibits M-CSF+RANKL-Induced Human Osteoclast Differentiation by Modulating NFATc1.

Parathyroid hormone-related protein (PTHrP) C-terminal peptides regulate the metabolism of bone cells. PHTrP [107–111] (osteostatin) promotes bone repair in animal models of bone defects and prevents bone erosion in inflammatory arthritis. In addition to its positive effects on osteoblasts, osteostatin may inhibit bone resorption. The aim of this study was to determine the effects of osteostatin on human osteoclast differentiation and function. We used macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor κB ligand (RANKL) to induce the osteoclast differentiation of adherent human peripheral blood mononuclear cells. Tartrate-resistant acid phosphatase (TRAP) staining was performed for the detection of the osteoclasts. The function of mature osteoclasts was assessed with a pit resorption assay. Gene expression was evaluated with qRT-PCR, and nuclear factor of activated T cells, cytoplasmic 1 (NFATc1) nuclear translocation was studied by immunofluorescence. We observed that osteostatin (100, 250 and 500 nM) decreased the differentiation of osteoclasts in a concentration-dependent manner, but it did not modify the resorptive ability of mature osteoclasts. In addition, osteostatin decreased the mRNA levels of cathepsin K, osteoclast associated Ig-like receptor (OSCAR) and NFATc1. The nuclear translocation of the master transcription factor in osteoclast differentiation NFATc1 was reduced by osteostatin. Our results suggest that the anti-resorptive effects of osteostatin may be dependent on the inhibition of osteoclastogenesis. This study has shown that osteostatin controls human osteoclast differentiation in vitro through the downregulation of NFATc1.

Treatment with DC/AML Fusion Vaccine and CD3xCD123 Bi-Specific T-Cell Engager (CD123-CODV-TCE) for Treatment of Acute Myeloid Leukemia

Treatment with DC/AML Fusion Vaccine and CD3xCD123 Bi-Specific T-Cell Engager (CD123-CODV-TCE) for Treatment of Acute Myeloid Leukemia

An Explorative Study on Monocyte Reprogramming in the Context of Periodontitis In Vitro and In Vivo.

AimsPeriodontitis is an independent risk factor for cardiovascular disease, but the mechanistic link is not fully understood. In atherosclerotic cardiovascular disease, monocytes can adopt a persistent hyperresponsive phenotype, termed trained immunity. We hypothesized that periodontitis-associated bacteria can induce trained immunity in monocytes, which subsequently accelerate atherosclerosis development.Materials and MethodsWe combined in vitro experiments on human primary monocytes and in vivo techniques in patients with periodontitis to test this hypothesis. Adherent peripheral blood mononuclear cells (PBMCs) were transiently exposed in vitro to Porphyromonas gingivalis for 24 hours, and restimulated with lipopolysaccharide (LPS) or Pam3CysK4 (P3C) six days later, to measure interleukin-6 (IL-6) and tumor necrosis factor α (TNFα) production. In an exploratory observational study, patients with severe periodontitis (63 ± 6 years, n=14) and control subjects with no-to-mild periodontitis (54 ± 10 years, n=14) underwent venipuncture and 2’-deoxy-2’-[18F]fluoro-D-glucose positron-emission-tomography ([18F]FDG PET/CT) scanning.ResultsWhen adherent peripheral blood mononuclear cells (PBMCs) were transiently exposed in vitro to Porphyromonas gingivalis for 24 hours, and restimulated with LPS or P3C six days later, IL-6 and TNFα production was significantly increased (TNFα/P3C, p<0.01). Circulating leukocytes, IL-6 and interleukin-1 receptor antagonist (IL-1Ra) concentrations were generally higher in patients compared to controls (leukocytes: p<0.01; IL-6: p=0.08; IL-1Ra: p=0.10). Cytokine production capacity in PBMCs after 24h stimulation revealed no differences between groups. [18F]FDG PET/CT imaging showed a trend for increased [18F]FDG-uptake in the periodontium [mean standard uptake value (SUVmean), p=0.11] and in femur bone marrow (SUVmean, p=0.06), but no differences were observed for vascular inflammation. Positive correlations between severity of periodontitis, measured by The Dutch Periodontal Screening Index and pocket depth, with circulating inflammatory markers and tissue inflammation were found.ConclusionsP. gingivalis induces long-term activation of human monocytes in vitro (trained immunity). Patients with severe periodontitis did have signs of increased systemic inflammation and hematopoietic tissue activation. However, their circulating monocytes did not show a hyperresponsive phenotype. Together we suggest that trained immunity might contribute to local periodontal inflammation which warrants further investigation.

Efficacious Dendritic Cell Based Immunotherapy for Advanced Solid Tumors: Three Case Studies

Background and Aims: Dendritic cell (DC)-based immunotherapy is promising as a viable tool in cancer treatment. Dendritic cell (DC)-based vaccination can provoke antitumor T cell responses in vivo. These case studies examined feasibility and outcome of DC-based tumor vaccination for patients with advanced cancers. This approach has been used mostly in patients in the presence of defined tumor antigens. Experimental Design: Accessible tumor tissue was disrupted into single cell suspensions. Autologous DCs were prepared from adherent peripheral blood mononuclear cells and cultured in granulocyte macrophage colony-stimulating factor, interleukin and autologous plasma. Tumor cells and DCs were co-cultured in the presence of polyethylene glycol to generate the fusions. Fusion cells were quantified by determining the percentage of cells that co-express tumor and DC markers. Patients were vaccinated with three doses of DC (10 X 106 ) were administered after every 2 weeks’ intervals and assessed weekly for toxicity, and tumor response was assessed at 3 months after completion of vaccination. Results: Vaccination was well tolerated. No physical signs of autoimmunity were detected. There was no evidence of significant toxicity from vaccine or adjuvant. There was increased expression of T helper type 1 cytokines. Vaccination resulted in a significant reduction in the level of prostate-specific antigen (PSA) in the prostate cancer patient. Disease was stable upto 6 months in cases of breast and cervical cancer patients. Conclusion: DC-based vaccination can stimulate an antitumoral T cell response in patients with various cancers. This data indicates that vaccination with autologous tumor-pulsed DCs generated from peripheral blood is safe and can induce tumorspecific cellular cytotoxicity. Clinical responses are achievable, even in patients with advanced disease.

Infectivity and viability of dengue virus infected hepatocytes cocultured with peripheral blood mononuclear cells from a healthy subject

BACKGROUND Dengue virus (DENV) can infect and replicate in monocytes, resulting in antibody-dependent enhancement. The liver is the main target of DENV, and the infection mechanisms of DENV include direct cytopathic effects (CPEs) of the virus, mitochondrial dysfunction, and effect of cellular and humoral immune factors in the liver. This study was aimed to explore the infectivity of DENV and viability of human hepatocytes using Huh 7it-1 cells cocultured with peripheral blood mononuclear cells (PBMCs).&#x0D; METHODS Huh 7it-1 cells were infected with dengue virus serotype-2 (DENV-2) New Guinea C strain at multiplicity of infection of 0.5 and 1 FFU/cell, and cocultured in vitro with and without adherent PBMCs. The infectivity of DENV was assessed by immunoperoxidase staining. The viability of Huh 7it-1 cells was assessed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, a tetrazole) assay and trypan blue staining. Data were statistically analyzed by Shapiro–Wilk and analysis of variance for normality significances.&#x0D; RESULTS The result showed that addition of PBMCs to DENV-2 infected Huh 7it-1 cells decreased the infectivity of DENV (15–37%). DENV-2 infection decreased the viability of Huh 7it-1 cells (15.5–20.8%). Despite the decrease in infectivity of DENV, the addition of PBMCs increased the Huh 7it-1 cells viability (4.5–10.2%).&#x0D; CONCLUSIONS Addition of PBMCs to Huh 7it-1 cells that are infected with DENV-2 decreased the infectivity of DENV and increased Huh 7it-1 cells viability.

Design of Artificial Immunogens Containing Melanoma-associated T-cell Epitopes.

Immunotherapy based on induction of T-cell responses is a promising approach to cancer treatment. The study aims to design artificial epitope-based immunogens, DNA vaccine candidates against melanoma and evaluate their ability to stimulate tumor cytotoxicity of ex vivo generated T-cells. The original computational methods were used for predicting T-cell epitopes and designing polyepitope melanoma antigens. Artificial genes encoding the target antigens were cloned into DNA vaccine plasmid vector. Target gene expression was confirmed both at transcriptional and translational level in HEK-293T cells transfected with DNA-vaccine constructs. Dendritic cells were generated from adherent peripheral blood mononuclear cells of HLA-A*02:01+ donors. Cytotoxic activity of effector lymphocytes stimulated in co-culture with autologous antigen-presenting dendritic cells towards melanoma Mel Is cells was assessed with lactate dehydrogenase release assay. The proportion of granzyme B producing CD8+ T-cells was estimated using intracellular cytokine staining and flow cytometry. Two DNA vaccine constructions were created - pMEL-TCI and pMEL-A0201 - encoding polypeptides containing T-cell epitopes of six immunodominant melanoma antigens (NY-ESO-1, MART1, MAGE-A1, MAGE-A11, MAGE-A3, and MAGE-C1). Dendritic cells transfected with DNA vaccine constructs were found to stimulate both tumor cytotoxicity mediated by autologous lymphocytes and granzyme B production by CD8+ T-cells, and pMEL-A0201 was found to be the most efficient. The described approach may become a common platform for designing immunotherapeutic vaccines against oncological diseases.

Inflammatory Response of Human Peripheral Blood Mononuclear Cells and Osteoblasts Incubated With Metallic and Ceramic Submicron Particles.

Inflammatory reactions associated with osteolysis and aseptic loosening are the result of wear particles generated at the articulating surfaces of implant components. The aim of the present study was to analyze the biological response of human osteoblasts and peripheral blood mononuclear cells (PBMCs) after exposure to metallic and alumina ceramic particles regarding cellular differentiation, cytokine release, and monocyte migration. Cells were exposed to particles (0.01 and 0.05 mg/ml) from an alumina matrix composite (AMC) ceramic and a CoCr28Mo6 alloy with an average size of 0.5 µm over 48 and 96 h. The expression rates of osteogenic (Col1A1, ALP) and pro-osteoclastic (RANK, Trap5b) differentiation markers as well as pro-osteolytic mediators (MMP-1, TIMP-1, IL-6, IL-8, MCP-1) were determined and soluble protein concentrations of active MMP-1, IL-6, IL-8, and pro-collagen type 1 in cell culture supernatants were evaluated. Additionally, the capacity of particle-treated osteoblasts to attract potentially pro-inflammatory cells to the site of particle exposure was investigated by migration assays using osteoblast-conditioned media. The cellular morphology and metabolism of human osteoblasts and adherent PBMCs were influenced by particle type and concentration. In human osteoblasts, Col1A1 expression rates and protein production were significantly reduced after exposing cells to the lower concentration of cobalt-chromium (CoCr) and AMC particles. Exposure to AMC particles (0.01 mg/ml) resulted in increased mRNA levels of RANK and Trap5b in adherent PBMCs. For MMP-1 gene expression, elevated levels were more prominent after incubation with CoCr compared to AMC particles in osteoblasts, which was not reflected by the protein data. Interleukin (IL)-6 and IL-8 mRNA and protein were induced in both cell types after treatment with AMC particles, whereas exposure to CoCr particles resulted in significantly upregulated IL-6 and IL-8 protein contents in PBMCs only. Exposure of osteoblasts to CoCr particles reduced the chemoattractant potential of osteoblast-conditioned medium. Our results demonstrate distinct effects of AMC and CoCr particles in human osteoblasts and PBMCs. Complex cell and animal models are required to further evaluate the impact of cellular interactions between different cell types during particle exposure.

Vaccine-Mediated Mechanisms Controlling Replication of Francisella tularensis in Human Peripheral Blood Mononuclear Cells Using a Co-culture System.

Cell-mediated immunity (CMI) is normally required for efficient protection against intracellular infections, however, identification of correlates is challenging and they are generally lacking. Francisella tularensis is a highly virulent, facultative intracellular bacterium and CMI is critically required for protection against the pathogen, but how this is effectuated in humans is poorly understood. To understand the protective mechanisms, we established an in vitro co-culture assay to identify how control of infection of F. tularensis is accomplished by human cells and hypothesized that the model will mimic in vivo immune mechanisms. Non-adherent peripheral blood mononuclear cells (PBMCs) were expanded with antigen and added to cultures with adherent PBMC infected with the human vaccine strain, LVS, or the highly virulent SCHU S4 strain. Intracellular numbers of F. tularensis was followed for 72 h and secreted and intracellular cytokines were analyzed. Addition of PBMC expanded from naïve individuals, i.e., those with no record of immunization to F. tularensis, generally resulted in little or no control of intracellular bacterial growth, whereas addition of PBMC from a majority of F. tularensis-immune individuals executed static and sometimes cidal effects on intracellular bacteria. Regardless of infecting strain, statistical differences between the two groups were significant, P < 0.05. Secretion of 11 cytokines was analyzed after 72 h of infection and significant differences with regard to secretion of IFN-γ, TNF, and MIP-1β was observed between immune and naïve individuals for LVS-infected cultures. Also, in LVS-infected cultures, CD4 T cells from vaccinees, but not CD8 T cells, showed significantly higher expression of IFN-γ, MIP-1β, TNF, and CD107a than cells from naïve individuals. The co-culture system appears to identify correlates of immunity that are relevant for the understanding of mechanisms of the protective host immunity to F. tularensis.

The Impact of Metal Ion Exposure on the Cellular Behavior of Human Osteoblasts and PBMCs: In Vitro Analyses of Osteolytic Processes.

Osteolysis in the periprosthetic tissue can be caused by metallic wear particles and ions that can originate from implant surface corrosion. These products influence cellular behavior and stimulate the expression of proinflammatory cytokines. The purpose of this study was to evaluate the impact of CoCr29Mo6 ions on cell survival, differentiation, and cytokine expression in human osteoblasts and peripheral blood mononuclear cells (PBMCs). Thus, we exposed cells with a mixture of 200 µg/L ion solution and determined cell viability and apoptosis/necrosis. Gene expression analyses of osteoblastic and osteoclastic differentiation markers as well as pro-osteolytic mediators (IL-6, IL-8, TNF-α, MCP-1, MMP1, TIMP1) were performed. These markers were also investigated in mixed cultures of adherent and non-adherent PBMCs as well as in co-cultures of human osteoblasts and PBMCs. The ion solution induced necrosis in osteoblasts and PBMCs in single cultures. All examined mediators were highly expressed in the co-culture of osteoblasts and PBMCs whereas in the single cell cultures only IL-6, IL-8, and MMP1 were found to be stimulated. While the applied concentration of the CoCr29Mo6 ion solutions had only marginal effects on human osteoblasts and PBMCs alone, the co-culture may provide a comprehensive model to study osteolytic processes in response to Co and Cr ions.

Exogenous alpha 1-antitrypsin down-regulates SERPINA1 expression.

The main goal of the therapy with purified human plasma alpha1-antitrypsin (A1AT) is to increase A1AT levels and to prevent lungs from elastolytic activity in patients with PiZZ (Glu342Lys) A1AT deficiency-related emphysema. Potential hepatic gains of this therapy are unknown. Herein, we investigated the effect of A1AT therapy on SERPINA1 (gene encoding A1AT) expression. The expression of SERPINA1 was determined in A1AT or A1AT plus Oncostatin M (OSM) treated primary human hepatocytes isolated from liver tissues from A1AT deficient patients and control liver tissues. In addition, SERPINA1 mRNA was assessed in lung tissues from PiZZ emphysema patients with and without A1AT therapy, and in adherent human peripheral blood mononuclear cells (PBMC) isolated from healthy PiMM donors. In a dose-dependent manner purified A1AT lowered SERPINA1 expression in hepatocytes. This latter effect was more prominent in hepatocytes stimulated with OSM. Although it did not reach statistical significance (P = 0.0539)—analysis of lung tissues showed lower SERPINA1 expression in PiZZ emphysema patients receiving augmentation therapy relative to those without therapy. Finally, exogenously added purified A1AT (1mg/ml) reduced SERPINA1 expression in naïve as well as in lipopolysaccharide (LPS)-stimulated human adherent PBMCs. Exogenous A1AT protein reduces its own endogenous expression. Hence, augmentation with native M-A1AT protein and a parallel reduction in expression of dysfunctional mutant Z-A1AT may be beneficial for PiZZ liver, and this motivates further studies.

The potentiating effect of hTFPI in the presence of hCD47 reduces the cytotoxicity of human macrophages.

In pig-to-human xenotransplantation, hyperacute rejection of pig organs could be overcome by the production of α1,3-galactosyltransferase knockout pigs. However, macrophage-mediated acute rejection is another obstacle that needs to be overcome. Among the various candidate genes involved in acute rejection, CD47 inhibits monocyte/macrophage-mediated phagocytosis by identifying the CD47 signal regulatory protein alpha (SIRP-α) as self/non-self. Tissue factor pathway inhibitor (TFPI) is involved in the regulation of the coagulation pathway and is able to bind to another ligand of CD47, thrombospondin-1 (TSP-1). When TSP-1 binds to CD47, phagocytosis in macrophages is increased. The 2A peptide system was used to establish pig kidney cells (PK15) simultaneously expressing human CD47 and human TFPI, and they were cultured with activated THP-1 cells. After staining with 7-aminoactinomycin D, flow cytometry analysis was carried out. TFPI siRNA analysis and recombinant human TFPI (rhTFPI) treatment were performed to determine the potentiating effect of TFPI on pig cells for activated THP-1 cells in the presence of CD47. Related inflammatory cytokines produced by activated THP-1 cells were analyzed using qPCR and Western blot technique. In addition, the tyrosine phosphorylation level of SIRP-α in activated THP-1 cells was analyzed using immunoprecipitation and Western blot. hCD47/hTFPI-PK15 cells survived better than hCD47-PK15, hTFPI-PK15, or normal PK15 cells on cytotoxicity tests using activated THP-1 cells. TSP-1, derived from these activated THP-1 cells, served as a mediator for this enhancing effect, and it also played a role in activated adherent peripheral blood mononuclear cells (PBMCs). The tyrosine phosphorylation level of SIRP-α in activated THP-1 cells was further increased in the case of co-expression of CD47/TFPI than in individual non-expression or expression of CD47 or TFPI alone. When hCD47 was expressed, the expression of hTFPI leaded to tyrosine phosphorylation of SIRP-α in activated THP-1 cells via hTSP-1 inhibition, and consequently, it might improve the effect of hCD47-SIRP-a signaling.

DC/Aml Fusion Cell Vaccination Administered to AML Patients Who Achieve a Complete Remission Potently Expands Leukemia Reactive T Cells and Is Associated with Durable Remissions

We have developed an autologous whole cell leukemia vaccine, whereby patient derived leukemic blasts are fused to ex-vivo generated dendritic cells. In this way, a broad array of leukemia-associated antigens are presented in the context of DC mediated co-stimulation, resulting in the expansion of a polyclonal anti-leukemia immune response. We are conducting a clinical trial in which AML patients who are not candidates for allogeneic transplantation undergo vaccination with DC/AML fusion cells after achieving a complete remission. 56 patients underwent collection of AML cells for vaccine generation from either a bone marrow aspirate (N=38), 20cc of peripheral blood (N=15) or a leukapheresis product (N=3) at presentation with newly diagnosed (N=55) or first relapsed (N=1) AML. The mean yield of AML cells was 692 x 106 cells with a mean viability of 99%. Patients who did not achieve remission or who underwent allogeneic transplant in first remission came off study and did not complete vaccine generation. Eligible patients achieving CR who did not proceed to allogeneic transplant (N=21) underwent leukapheresis for DC generation and vaccine preparation. Adherent peripheral blood mononuclear cells were cultured in the presence of GM-CSF and IL-4 for 5-7 days, and exposed to TNFα for 48-72 hours to generate mature DCs. Fusion cells were generated by co-culture of DCs with AML cells in the presence of 50% polyethylene glycol and identified as cells co-expressing antigens that were unique to the DC and tumor population. Mean fusion efficiency and viability was 42% and 87%, respectively. Vaccination with DC/leukemia fusion cells was initiated within 12 weeks from count recovery following the final cycle of post-remission therapy. 16 patients (median age 60) have received at least two monthly vaccinations at a dose of 5x106 fusion cells. Remission induction therapy consisted of 7+3 in 13 patients, 7+3 followed by 5+2 re-induction in 1 patient, MEC in one patient, and decitabine in 1 patient. 9 patients had intermediate risk cytogenetics, 2 patients had good risk cytogenetics, and 5 patients had poor cytogenetic risk disease. Vaccination was well tolerated, and importantly, was not associated with clinically significant auto-immunity. Possibly related adverse events were transient and of grade 1-2 intensity, including vaccine site reactions, pruritis, arthalgias, myalgias, eosinophilia, leukopenia, thrombocytopenia. Biopsy of vaccine site reactions demonstrated a dense infiltrate of CD4 and CD8 T cells consistent with recruitment of reactive T cell populations to the vaccine bed. To date, 12 of 16 evaluable patients remain in remission (75%), with a median follow up of 45 months. Notably, no patient has relapsed later than 1 year from completing chemotherapy and the patient who was treated in CR2 remains in remission with 51 months of follow up, after having relapsed within a year of CR1. Vaccination resulted in the potent induction of leukemia specific immunity as measured by a mean fold increase of 4.9 CD8 T cells expressing IFNγ in response to ex vivo exposure to autologous leukemia cell lysates (n=14). In a subset of patients who are HLA2.1+, vaccination resulted in the expansion of T cells recognizing MUC1 (4.4 fold increase), WT1 (2.7 fold increase), NY-ESO (3.8 fold increase) tumor antigens by pentamer analysis. In conclusion, DC/AML fusion cell vaccination administered to patients who achieve complete remission results in the potent expansion of leukemia reactive T cells, and durable remissions. A second cohort is planned in which patients will be treated with DC/AML fusion cell vaccination in conjunction with immune checkpoint blockade. DisclosuresStone:Karyopharm: Consultancy; Roche/Genetech: Consultancy; Merck: Consultancy; Novartis: Research Funding; Celgene: Consultancy; Pfizer: Consultancy; Agios: Consultancy; Sunesis: Consultancy, Other: DSMB for clinical trial; Amgen: Consultancy; Abbvie: Consultancy; Celator: Consultancy; AROG: Consultancy; Juno: Consultancy. Steensma:Celgene: Consultancy; Amgen: Consultancy; Incyte: Consultancy; Onconova: Consultancy. DeAngelo:Incyte: Consultancy; Celgene: Consultancy; Agios: Consultancy; Novartis: Consultancy; Ariad: Consultancy; Bristol Myers Squibb: Consultancy; Amgen: Consultancy; Pfizer: Consultancy. Kufe:Genus Oncology: Consultancy, Equity Ownership.

PD-L1 on Antigen-Presenting Cells Facilitate the Induction of Antigen-Specific Cytotoxic T Lymphocytes

Programmed death-ligand 1 (PD-L1) binds to programmed death-1 (PD-1) on T cells and contributes to T cell exhaustion during chronic infections. Moreover, it has been shown that engagement of PD-1 on T cells by PD-L1 on tumor cells is associated with the immune escape of tumors, and recent clinical trials have highlighted the anti-tumor efficacy of blockade of the PD-1/PD-L1 pathway. Along with costimulatory molecules such as CD80 and CD86, PD-L1 is also highly expressed on dendritic cells (DCs), which are professional antigen-presenting cells (APCs) that are widely used to generate antigen-specific cytotoxic T lymphocytes (CTLs) for adoptive immunotherapy. However, the roles of PD-L1 on APCs have not been well examined. Thus, we evaluated the roles of PD-L1 on APCs in the induction of cytomegalovirus (CMV)-specific CTLs (CMV-CTLs).First, peripheral blood mononuclear cells (PBMCs) were obtained from five CMV-seropositive healthy donors (HLA-A*02:01, n = 3; A*24:02, n = 1; B*07:02, n = 1). Mature DCs were generated from adherent PBMCs by stimulation with GM-CSF/IL-4 and maturational cytokines (TNF-a, IL-1b, IL-6, and PGE2), and freshly isolated CD3+ T cells were stimulated with mature DCs pulsed with CMV pp65-derived HLA-restricted peptides (NLVPMVATV for HLA-A*02:01, QYDPVAALF for A*24:02, and TPRVTGGGAM for B*07:02) in the presence or absence of anti-PD-L1 blocking antibody. After 14 days of culture, the presence of anti-PD-L1 antibody resulted in a less efficient induction of CMV-CTLs (Figure 1), suggesting that PD-L1 might play a positive role in the induction of antigen-specific CTLs. For further evaluation, we generated K562-based artificial APCs (aAPCs), which were retrovirally transduced with HLA class I molecules and various combinations of CD80/86 and PD-L1 (named K562, K562+CD80/86, K562+PD-L1, and K562+CD80/86+PD-L1). CD3+ T cells isolated from nine CMV-seropositive healthy donors (HLA-A*02:01, n = 4; A*24:02, n = 4; B*07:02, n = 1) were stimulated weekly for 28 days using HLA-restricted CMV peptide-pulsed K562-based aAPCs. K562+CD80/86+PD-L1 led to significantly higher induction of CMV-CTLs than K562 or K562+CD80/86 (Figure 2). Since the original K562 cells slightly express CD80, we completely knocked out the CD80 expression in the cells using the CRISPR/Cas9 system, and then we transduced the cells with or without PD-L1 (named CD80KO K562+PD-L1 and CD80KO K562, respectively) to determine whether PD-L1 itself has a stimulatory effect on the induction of CMV-CTLs. CD80KO K562 and CD80KO K562+PD-L1 induced the same amount of CMV-CTLs, suggesting that PD-L1 itself does not have a stimulatory effect for the induction of CMV-CTLs. Phenotypic analysis showed that CMV-CTLs induced by K562+CD80/86+PD-L1 contained more CD45RA- CD62L+ central memory T cells, which persist longer and are more effective in vivo after adoptive transfer in CTL therapy, than CMV-CTLs induced by other K562-based aAPCs. In addition, when compared with CMV-CTLs induced by other K562-based aAPCs, CMV-CTLs induced by K562+CD80/86+PD-L1 did not express higher levels of exhaustion markers such as PD-1 and TIM-3, and exhibited similar levels of IFN-gamma and IL-2 production and CD107a expression in response to the specific peptide stimulation. Furthermore, to evaluate the effect of K562-based aAPCs on the induction of tumor antigen-specific CTLs, CD3+ T cells were isolated from three HLA-A*24:02-positive healthy donors and stimulated with HLA-A*24:02-restricted WT1 peptide-pulsed K562-based aAPCs weekly. After four stimulations, only K562+CD80/86+PD-L1 could clearly expand the WT1-specific CTLs from all of the three donors (Figure 3).In conclusion, our findings demonstrate that PD-L1 expressed on APCs along with CD80/86 enhanced the induction of antigen-specific CTLs without causing excessive differentiation or functional exhaustion of the induced CTLs. These results suggest that stimulation with K562+CD80/86 might result in the activation-induced cell death of antigen-specific CTLs and that PD-L1 might be involved in fine-tuning excessive stimulation of CD80/86. Further analyses to determine the mechanisms of our findings are warranted. Our results also highlight the possibility that K562+CD80/86+PD-L1 has therapeutic potential as novel aAPCs for the generation of CTLs for adoptive immunotherapy. [Display omitted] DisclosuresKiyoi:Takeda Pharmaceutical Co., Ltd.: Research Funding; Pfizer Inc.: Research Funding; Teijin Ltd.: Research Funding; Taisho Toyama Pharmaceutical Co., Ltd.: Research Funding; Eisai Co., Ltd.: Research Funding; Yakult Honsha Co.,Ltd.: Research Funding; MSD K.K.: Research Funding; Alexion Pharmaceuticals: Research Funding; Novartis Pharma K.K.: Research Funding; Astellas Pharma Inc.: Consultancy, Research Funding; Japan Blood Products Organization: Research Funding; Nippon Shinyaku Co., Ltd.: Research Funding; FUJIFILM RI Pharma Co.,Ltd.: Research Funding; Nippon Boehringer Ingelheim Co., Ltd.: Research Funding; FUJIFILM Corporation: Patents & Royalties, Research Funding; Zenyaku Kogyo Co., Ltd.: Research Funding; Sumitomo Dainippon Pharma Co., Ltd.: Research Funding; Kyowa Hakko Kirin Co., Ltd.: Consultancy, Research Funding; Bristol-Myers Squibb: Research Funding; Chugai Pharmaceutical Co., Ltd.: Research Funding; Mochida Pharmaceutical Co., Ltd.: Research Funding.

Peripheral Blood Mononuclear Cells Enhance Cartilage Repair in in vivo Osteochondral Defect Model.

This study characterized peripheral blood mononuclear cells (PBMC) in terms of their potential in cartilage repair and investigated their ability to improve the healing in a pre-clinical large animal model. Human PBMCs were isolated with gradient centrifugation and adherent PBMC’s were evaluated for their ability to differentiate into adipogenic, chondrogenic and osteogenic lineages and also for their expression of musculoskeletal genes. The phenotype of the PBMCs was evaluated using Stro-1, CD34, CD44, CD45, CD90, CD106, CD105, CD146 and CD166 cell surface markers. Osteochondral defects were created in the medial femoral condyle (MFC) of 24 Welsh mountain sheep and evaluated at a six month time point. Four cell treatment groups were evaluated in combination with collagen-GAG-scaffold: (1) MSC alone; (2) MSCs and PBMCs at a ratio of 20:1; (3) MSCs and PBMC at a ratio of 2:1 and (4) PBMCs alone. Samples from the surgical site were evaluated for mechanical properties, ICRS score and histological repair. Fresh PBMC samples were 90% positive for hematopoietic cell surface markers and negative for the MSC antibody panel (<1%, p = 0.006). However, the adherent PBMC population expressed mesenchymal stem cell markers in hypoxic culture and lacked CD34/45 positive cells (<0.2%). This finding demonstrated that the adherent cells had acquired an MSC-like phenotype and transformed in hypoxia from their original hematopoietic lineage. Four key genes in muskuloskeletal biology were significantly upregulated in adherent PBMCs by hypoxia: BMP2 4.2-fold (p = 0.0007), BMP6 10.7-fold (p = 0.0004), GDF5 2.0-fold (p = 0.002) and COL1 5.0-fold (p = 0.046). The monolayer multilineage analysis confirmed the trilineage mesenchymal potential of the adherent PBMCs. PBMC cell therapy was equally good as bone marrow MSC therapy for defects in the ovine large animal model. Our results show that PBMCs support cartilage healing and oxygen tension of the environment was found to have a key effect on the derivation of a novel adherent cell population with an MSC-like phenotype. This study presents a novel and easily attainable point-of-care cell therapy with PBMCs to treat osteochondral defects in the knee avoiding any cell manipulations outside the surgical room.

Autologous tumor lysate-loaded dendritic cell vaccination combined with Sunitinib for metastatic renal cell carcinoma

Background The antibodies that block immunological checkpoints have been successfully applied for the treatment of cancer. Similar effects are expected by molecular targeted therapy, which primarily aims to inhibit molecular pathways for tumor cell growth and survival. Such small molecular drugs may modulate the immune system, which raises the possibility that targeted therapy might be effectively combined with immunotherapy. Sunitinib, a tyrosine kinase inhibitor currently in use for the treatment of metastatic renal cell carcinoma (mRCC), has been reported to modulate immunosuppressive cells such as myeloid-derived suppressor cells (MDSCs) and regulatory T cells (Tregs). We conducted a clinical study of dendritic cell (DC)-based immunotherapy together with Sunitinib in mRCC patients in an effort to enhance immunotherapeutic efficacy by inhibiting immunosuppressive cells. Methods Patients aged ≥20 years with advanced or recurrent mRCC who underwent nephrectomy were eligible for this study. Autologous tumor samples were obtained by surgery and used for preparing autologous tumor lysate. Leukapheresis was performed to obtain peripheral blood mononuclear cells (PBMCs). DCs were generated from adherent PBMCs in the presence of recombinant human granulocyte macrophage colony-stimulating factor (GM-CSF) (500 IU/ml) and IL-4 (500 IU/ml). Mature DCs were loaded with autologous tumor lysate by electroporation. Eight patients were enrolled in the study and received Sunitinib at a dose of 50 mg p.o. daily for 28 days followed by 14 days of rest. Tumor lysate-loaded DCs were administered subcutaneously every two weeks, with concomitant Sunitinib. Results

A pilot study of autologous tumor lysate-loaded dendritic cell vaccination combined with sunitinib for metastatic renal cell carcinoma.

BackgroundSunitinib, a tyrosine kinase inhibitor currently in use for the treatment of metastatic renal cell carcinoma (mRCC), has been reported to modulate immunosuppressive cells such as myeloid-derived suppressor cells (MDSCs) and regulatory T cells (Tregs) in addition to exerting anti-angiogenic effects. We conducted a clinical trial of dendritic cell (DC)-based immunotherapy together with sunitinib in mRCC patients in an effort to enhance immunotherapeutic efficacy by inhibiting immunosuppressive cells.MethodsPatients aged ≥20 years with advanced or recurrent mRCC who underwent nephrectomy were eligible for this study. Autologous tumor samples were obtained by surgery under aseptic conditions and used for preparing autologous tumor lysate. About 4 weeks after surgery, leukapheresis was performed to isolate peripheral blood mononuclear cells (PBMCs). DCs were generated from adherent PBMCs in the presence of recombinant human granulocyte macrophage colony-stimulating factor (GM-CSF) (500 IU/ml) and recombinant human IL-4 (500 IU/ml). Autologous tumor lysate was loaded into mature DC by electroporation. Eight patients were enrolled in the study and received sunitinib at a dose of 50 mg p.o. daily for 28 days followed by 14 days of rest. Tumor lysate-loaded DCs were administered subcutaneously every two weeks, with concomitant sunitinib.ResultsNo severe adverse events related to vaccination were observed. Sunitinib decreased the frequencies of MDSCs in peripheral blood of 5 patients and of Tregs in 3. Tumor lysate-reactive CD4 or CD8 T cell responses were observed in 5 patients, 4 of whom showed decreased frequencies of Tregs and/or MDSCs. The remaining 3 patients who failed to develop tumor-reactive T cell responses had high levels of IL-8 in their sera and did not show consistent reductions in MDSCs and Tregs.ConclusionsDC-based immunotherapy combined with sunitinib is safe and feasible for patients with mRCC.Trial registrationUMIN000002136

Ivermectin-dependent attachment of neutrophils and peripheral blood mononuclear cells to Dirofilaria immitis microfilariae in vitro

The macrocyclic lactones are the only anthelmintics used to prevent heartworm disease, but it is very difficult to reproduce their in vivo efficacy against Dirofilaria immitis larvae in experiments in vitro. These assays typically measure motility, suggesting that paralysis is not the mode of action of the macrocyclic lactones against D. immitis. We isolated peripheral blood mononuclear cells (PBMC) and neutrophils from uninfected dogs and measured their adherence to D. immitis microfilariae in the presence of varying concentrations of ivermectin. We found that adherence of PBMC to the microfilariae was increased in the presence of ivermectin concentrations ≥100nM and adherence of neutrophils was increased in drug concentrations ≥10nM. Up to 50% of microfilariae had adherent PBMC in the presence of the drug, and binding was maximal after 40h incubation. Neutrophil adherence was maximal after 16h, with approximately 20% of the microfilariae having at least one cell adhered to them. Adherent neutrophils showed morphological evidence of activation. These results are consistent with a model in which the macrocyclic lactones interfere with the parasites ability to evade the host's innate immune system.

Study of plasma-induced peripheral blood mononuclear cells survival using Fourier transform infrared microspectroscopy

Components present in the acellular fraction of blood influence the blood cell survival and function and the response to biotic and abiotic factors. Human plasma and sera have been used as therapeutic agents and are known to increase cell survival. White blood cells in normal blood are exposed to plasma components in vivo, but the effect of such plasma components in vitro on adherent peripheral blood mononuclear cells (PBMCs) that includes monocytes has not been fully investigated. We cultured human PBMCs with autologous plasma and observed structural variation due to plasma addition in PBMCs along with increased cell survival. Light microscopy of the cells showed increased granularity in plasma-treated cells. Fourier transform infrared (FTIR) spectroscopy was used to elucidate the possible mechanism by studying the changes in the biochemical composition of the cells that explained the observations. FTIR spectroscopy of plasma-treated cells show altered spectral pattern in the mid-IR region, indicating increased phospholipid levels. Heat-stable components in the plasma possibly increase the differentiation of PBMCs, as evident by increased phospholipid metabolism. The data suggest that plasma-stimulated membrane biogenesis may contribute to PBMC survival by inducing them to differentiate into antigen presenting cells (APCs) like macrophages and dendritic cells.

Clinical Trial Evaluating DC/AML Fusion Cell Vaccination In AML Patients

We have developed a promising leukemia vaccine in which patient derived AML cells are fused with autologous dendritic cells (DCs), presenting a broad array of antigens. We are conducting a clinical trial in which AML patients who are not candidates for allogeneic transplantation undergo vaccination with DC/AML fusion cells following chemotherapy induced remission. Twenty-six patients (14 males, 12 females) underwent collection of AML cells at disease presentation for vaccine generation and immune monitoring studies. Median age of the patients is 66 years. Tumor was collected from either a bone marrow aspirate (N=16), 20 cc of peripheral blood (N=7), or leukapheresis product (N=3) at the time of presentation with newly diagnosed AML (N=25) or first relapsed AML (N=1). The mean yield of AML cells was 109 x 106 cells with a mean viability of 91%. Eligible patients achieving CR following chemotherapy (N=16) underwent leukapheresis for DC generation and vaccine preparation. Adherent peripheral blood mononuclear cells were isolated, cultured in the presence of GM-CSF and IL-4 for 5-7 days, and exposed to TNFα for 48-72 hours to generate mature DCs. The mean yield of DCs was 177 x106 cells with a mean viability of 89%. Fusion cells were generated by co-culture of DCs with AML cells in the presence of 50% polyethylene glycol and identified as cells co-expressing antigens that were unique to the DC and tumor population. Mean fusion efficiency and viability was 38% and 85%, respectively. As a measure of their activity as antigen presenting cells, the capacity of fusion cells to stimulate allogeneic T cell proliferation ex vivo was quantified. In contrast to the leukemia preparation (mean stimulation index (SI) 3.81), the DC and fusion cell preparation were potent stimulators (mean SI 19.61 and 13.48, respectively). Vaccination with DC/leukemia fusion cells was initiated within 12 weeks from count recovery following the final cycle of chemotherapy. 13 patients received at least two monthly vaccinations at a dose of 5x106 fusion cells. 8 patients had intermediate risk cytogenetics, 3 patients had good risk cytogenetics, and 2 patients had a complex karyotype. Vaccination was well tolerated, and importantly, was not associated with clinically significant auto-immunity. Possibly related adverse events were transient and of grade 1-2 intensity, including vaccine site reactions, pruritis, arthalgias, myalgias, eosinophilia, leukopenia, thrombocytopenia. Biopsy of vaccine site reactions demonstrated a dense infiltrate of CD4 and CD8 T cells consistent with recruitment of reactive T cell populations to the vaccine bed. To date, 9 patients remain in remission (69%), with a mean follow up of 23 months. Peripheral blood samples were collected prior to each vaccination and at 1, 3, and 6 months following completion of vaccination. Vaccination resulted in the potent induction of leukemia specific immunity as measured by an increase in CD8 T cells expressing IFNγ in response to ex vivo exposure to autologous leukemia cell lysates (mean fold increase 8, n=6). Bone marrow derived T cells were isolated prior to and following vaccination in patients who are HLA2.1+. Vaccination resulted in the expansion of bone marrow infiltrating T cells recognizing MUC1 (9 fold increase), WT1 (5 fold increase), PRAME (12 fold increase) tumor antigens by tetramer analysis (n=2). In conclusion, DC/AML fusion cell vaccination results in the potent expansion of leukemia reactive T cells and durable remissions following chemotherapy. Enrollment to a second cohort is being initiated, in which patients with be treated with DC/AML fusion cell vaccination in conjunction with PD1 blockade. Disclosures:No relevant conflicts of interest to declare.

Decreased and dysfunctional circulating endothelial progenitor cells in patients with chronic obstructive pulmonary disease

Background It has been widely demonstrated that endothelial progenitor cells are involved in several diseases and that they have therapeutic implications. In order to define the altered pulmonary vascular homeostasis in chronic obstructive pulmonary disease, we sought to observe the level and functions of circulating endothelial progenitor cells in patients with chronic obstructive pulmonary disease. Methods The total study population included 20 patients with chronic obstructive pulmonary disease and 20 control subjects. The number of circulating endothelial progenitor cells (CD34+/CD133+/VEGFR-2+ cells) was counted by flow cytometry. Circulating endothelial progenitor cells were also cultured in vitro and characterized by uptake of DiIacLDL, combining with UEA-I, and expression of von Willebrand factor and endothelial nitric oxide synthase. Adhesion, proliferation, production of nitric oxide, and expression of endothelial nitric oxide synthase and phosphorylated-endothelial nitric oxide synthase were detected to determine functions of circulating endothelial progenitor cells in patients with chronic obstructive pulmonary disease. Results The number of circulating endothelial progenitor cells in the chronic obstructive pulmonary disease group was lower than in the control group: (0.54±0.16)% vs. (1.15±0.57)%, P &lt;0.05. About 80% of adherent peripheral blood mononuclear cells cultured in vitro were double labeled with Dil-acLDL and UEA-1. The 92% and 91% of them were positive for von Willebrand factor and endothelial nitric oxide synthase, respectively. Compared with the control, there were significantly fewer adhering endothelial progenitor cells in chronic obstructive pulmonary disease patients: 18.7±4.8/field vs. 45.0±5.9/field, P &lt;0.05. The proliferation assay showed that the proliferative capacity of circulating endothelial progenitor cells from chronic obstructive pulmonary disease patients was significantly impaired: 0.135±0.038 vs. 0.224±0.042, P &lt;0.05. Furthermore, nitric oxide synthase (112.06±10.00 vs. 135.41±5.38, P &lt;0.05), phosphorylated endothelial nitric oxide synthase protein expression (88.89±4.98 vs. 117.98±16.49, P &lt;0.05) and nitric oxide production ((25.11±5.27) μmol/L vs. (37.72±7.10) μmol/L, P &lt;0.05) were remarkably lower in endothelial cells from the chronic obstructive pulmonary disease group than the control. Conclusion Circulating endothelial progenitor cells were decreased and functionally impaired in patients with chronic obstructive pulmonary disease.