Nonadherent Peripheral Blood Mononuclear Cells Research Articles

Pancreatic beta cells persistently infected with coxsackievirus B4 are targets of NK cell-mediated cytolytic activity.

It has been suggested that the persistence of coxsackieviruses-B (CV-B) in pancreatic beta cells plays a role in the pathogenesis of type 1 diabetes (T1D). Yet, immunological effectors, especially natural killer (NK) cells, are supposed to clear virus-infected cells. Therefore, an evaluation of the response of NK cells to pancreatic beta cells persistently infected with CV-B4 was conducted. A persistent CV-B4 infection was established in 1.1B4 pancreatic beta cells. Infectious particles were found in supernatants throughout the culture period. The proportion of cells containing viral protein VP1 was low (< 5%), although a large proportion of cells harbored viral RNA (around 50%), whilst cell viability was preserved. HLA class I cell surface expression was downregulated in persistently infected cultures, but HLA class I mRNA levels were unchanged in comparison with mock-infected cells. The cytolytic activities of IL-2-activated non-adherent peripheral blood mononuclear cells (PBMCs) and of NK cells were higher towards persistently infected cells than towards mock-infected cells, as assessed by an LDH release assay. Impaired cytolytic activity of IL-2-activated non-adherent PBMCs from patients with T1D towards infected beta cells was observed. In conclusion, pancreatic beta cells persistently infected with CV-B4 can be lysed by NK cells, implying that impaired cytolytic activity of these effector cells may play a role in the persistence of CV-B in the host and thus in the viral pathogenesis of T1D.

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.

Mechanism of activation of natural killer and CD8 T cells by interleukin 15 in the prostate cancer microenvironment

BackgroundThe prostate cancer microenvironment profoundly inhibits effector immune-cell activity. We have shown that interleukin (IL) 15, unlike other cytokines proposed for anti-tumour immunotherapy such as IL12 and IL21, can increase natural killer (NK) cell and CD8 T-cell activity within the prostate cancer microenvironment. To investigate mechanisms of this activation, we studied the effects of IL15 on inhibitory and activatory receptors on NK cells and their corresponding ligands on prostate cancer cells. NK receptors known as killer-Ig-like receptors (KIRs) have a crucial role in NK-mediated killing of tumour cells. NK-receptor ligands on tumour cells can attenuate NK activity by binding their corresponding inhibitory receptors. Conversely, increased HLA-related ligand expression can increase the recognition and killing of tumour cells by T cells. MethodsThe prostate cancer cell-lines PC3 and LNCaP were incubated with non-adherent peripheral blood mononuclear cells (PBMCs) at effector-to-target ratios of 8:1 and cytokines IL2 or IL15. After 1 week, NK cells in the co-cultures were stained with antibodies to inhibitory KIRs (KIR2DL1, KIR2DL2, KIR3DL1) and activatory KIRs (NKp44, NKG2D, NKp46, DNAM1). Tumour cells in the co-cultures were stained with antibodies to inhibitory NK-receptor ligands HLA-class1-Bw4 and HLA-G, and activatory receptor ligands Nectin-2, and MICA/B. Antibody to HLA-ABC was also used. Staining was followed by flow cytometric analysis. Groups were compared with one-way ANOVA and post-hoc Newman-Keuls (five repeated experiments). FindingsIL15, but not IL2, inhibited expression of KIR2DL1 and KIR3DL1 by up to 50% (p<0·001) on NK cells in PBMC-prostate cancer cell co-cultures. NKG2D was increased up to 40% (p<0·001) in these cells with IL15 but not IL2. On tumour cells, IL15 decreased HLA-Class-1-Bw4 and HLA-G by 60% (p<0·001) and 75% (p<0·001), respectively. No significant effects were seen on the activatory-ligands whereas HLA-ABC expression was increased by over 65% (p<0·001). InterpretationOne mechanism by which IL15 increases prostate cancer killing by NK cells in PBMC-prosate cancer co-cultures is through upregulating activatory NKG2D and decreasing inhibitory KIR2DL1 and KIR3DL1 receptors. In addition, inhibitory ligands on prostate cancer cells are downregulated, suggesting strong shifts towards NK activation versus inhibition in the co-cultures. Increased HLA-ABC also favours stronger cytotoxic-T-cell recognition. FundingHeathside Trust, UK Medical Research Council, National Institute for Health Research, Prostate cancer UK. The research was funded and supported by the National Institute for Health Research (NIHR) Biomedical Research Centre based at Guy's and St Thomas' NHS Foundation Trust and King's College London.

Anti-tumor immunity of gene vaccine with nucleofection technology

ObjectiveTo observe enhancement of anti-tumor immunity by gene vaccine using nucleofection technologyMethodsThe technique of nucleofection was used to transfer effectively plasmid DNA into immature dendritic cells (iDCs); we studied immune responses regulated by DNA vaccine using real-time quantitative polymerase chain reaction (PCR) and western-blotting to optimize the follow-up lymphocyte activation. The anti-tumor capacity of lymphocytes primed by DCs was analyzed using lactate dehydrogenase with a non-radioactive cytotoxicity assayResultsHuman monocyte-derived dendritic cells (hMoDCs) were induced by interleukin (IL)-4 and granulocyte-macrophage colonystimulating factor (GM-CSF) in vitro from human monocytes for 5 or 6 days. DNA vaccine was transfected to iDCs with high transfection (35.73%) using nucleofection. Compared with the iDC group, the expression of Th1 cell cytokine IL-12, IL-18 and Th2 cell cytokine IL-4 increased after stimulation. CD86 and CD83 were upregulated compared with non-nucleofected groups 48 hours after nucleofection with DC-pVAX-PRA. The result of the cytotoxicity assay showed that DCs-pVAX-PRA primed non-adherent peripheral blood mononuclear cells (PBMCs) exhibit their highest cytotoxicity against target cells.ConclusionThe results show that DNA vaccine was transfected to iDC with high transfection efficiency using nucleofection, priming autologous lymphocytes for anti-tumor immunity by upregulated expression of co-stimulatory molecules, adhesion molecules and cytokines. These results provided a basis to explore the molecular mechanism of DNA vaccine in vivo.

Dendritic cell-based vaccination of patients with advanced pancreatic carcinoma: results of a pilot study.

Dendritic cell (DC)-based vaccination can induce antitumor T cell responses in vivo. This clinical pilot study examined feasibility and outcome of DC-based tumor vaccination for patients with advanced pancreatic adenocarcinoma. Tumor lysate of patients with pancreatic carcinoma was generated by repeated freeze-thaw cycles of surgically obtained tissue specimens. Patients were eligible for DC vaccination after recurrence of pancreatic carcinoma or in a primarily palliative situation. DC were generated from peripheral blood mononuclear cells (PBMC), loaded with autologous tumor lysate, stimulated with TNF-α and PGE(2) and injected intradermally. All patients received concomitant chemotherapy with gemcitabine. Disease response was the primary endpoint. Individual immunological responses to DC vaccination were analyzed by T cell-based immunoassays using pre- and post-vaccination samples of non-adherent PBMC. Twelve patients received DC vaccination and concomitant chemotherapy. One patient developed a partial remission, and two patients remained in stable disease. Median survival was 10.5 months. No severe side effects were observed. Tumor-reactive T cells could be detected prior to vaccination. DC vaccination increased the frequency of tumor-reactive cells in all patients tested; however, the degree of this increase varied. To quantify the presence of tumor-reactive T cells, stimulatory indices (SI) were calculated as the ratio of proliferation-inducing capacity of lysate-loaded versus -unloaded DC. The patient with longest overall survival of 56 months had a high SI of 6.49, indicating that the presence of a pre-vaccination antitumor T cell response might be associated with prolonged survival. Five patients survived 1 year or more. DC-based vaccination can stimulate an antitumoral T cell response in patients with advanced or recurrent pancreatic carcinoma receiving concomitant gemcitabine treatment.

CT-011, Anti-PD-1 Antibody, Enhances Ex-Vivo T Cell Responses to Autologous Dendritic/Myeloma Fusion Vaccine Developed for the Treatment of Multiple Myeloma.

Abstract 781We have developed a promising cancer vaccine in which autologous tumor cells are fused with dendritic cells (DCs) resulting in the presentation of tumor antigens in the context of DC mediated costimulation. In animal models, vaccination with fusion cells results in eradication of established tumor, and in clinical trials, both immunologic and clinical responses have been observed. However, response to vaccination may be muted by inhibitory pathways that blunt activated T cell responses. The PD-1/PDL-1 pathway is an important element contributing to tumor mediated immune suppression. In infectious disease models, upregulation of T cell expression of PD-1 is associated with an exhausted phenotype facilitating the development of chronic viral infection. In contrast, PD-1 blockade results in the restoration of functionally active T cells and clearance of infection. The PD-1/PDL-1 pathway is also being evaluated a as a central mechanism by which tumors escape host immunity. CT-011, is a humanized anti PD-1 antibody that is currently evaluated in Phase II studies for the treatment of hematological malignancies and solid tumors. In this study, we evaluated expression of PD-1 on T cells derived from patients with advanced hematologic malignancies, and PDL-1 expression on primary myeloma cells, ex-vivo generated dendritic cells, and DC/tumor fusion cells. We evaluated the effect of PD-1 blockade with CT-011 on T cell response to DC/tumor fusion cell stimulation in vitro. Tumor cells were obtained from bone marrow aspirates of patients with multiple myeloma. Nonadherent peripheral blood mononuclear cells obtained from patients with multiple myeloma and normal volunteers were cultured in RPMI supplemented with 10U/ml IL-2, and expression of PD-1 on CD4+ T cells was assessed by flow cytometric analysis . DCs were generated from adherent mononuclear cells cultured with rhIL-4, GM-CSF and TNFα and fused with tumor cells by coculture in 50% solution of polyethylene glycol. T cells were stimulated by DC/tumor fusions in the presence or absence of 5ug/ml CT-011. We demonstrate that PD-1 expression is markedly upregulated on T cells in patients with advanced multiple myeloma. As compared to a control population of normal volunteers in which mean levels of PD-1 expression was 6% (n=7), mean expression in patients with multiple myeloma was 20% (n=9). These findings suggest that upregulation of PD-1 expression may play a central role in tumor mediated immune suppression. Mean expression of PDL-1 was 91% on dendritic cells generated from adherent peripheral blood mononuclear cells obtained from normal volunteers (n=6), and 66% on patient derived myeloma cells (n=3). In addition, PDL-1 expression is found in greater than 90% of DC/tumor fusions (n=2), which potentially provides an inhibitory signal dampening fusion mediated immunologic response. We examined the effect of PD-1 blockade on T cell response to DC/tumor fusions ex vivo with different anti PD-1 antibodies including CT-011. DC/tumor fusions were co-cultured with autologous T cells alone or with antibodies against PD-1 Enhanced fusion mediated stimulation of T cells was noted particularly with CT-011, resulting in a greater than 5 fold increase in T cell proliferation. Interferon gamma secretion by CD4+ T cells in response to stimulation by DC/myeloma fusion cells was increased from 4% to 11% in the presence of CT-011. In addition, IL-10 secretion by CD4+ cells following DC/myeloma fusion stimulation decreased from 6.5% to 3.5% following PD-1 blocakde . In summary, we have demonstrated that PD-1 expression is increased in T cells of patients with hematologic malignancy, and CT-011, a PD-1 blocking antibody, enhances activated T cell responses following stimulation with a DC/tumor fusion vaccine. A clinical trial in which patients with multiple myeloma are treated with DC/myeloma fusions in conjunction with CT-011 following autologous transplantation is planned. Disclosures:Schickler:CureTech, Ltd.: Employment. Rotem-Yehudar:CureTech, Ltd.: Employment.

A Comparative Analysis of Immune Reconstitution Following Reduced Intensity Conditioning with CAMPATH-1H and Total Lymphoid Irradiation/Anti-Thymocyte Globulin Prior to Allogeneic Stem Cell Transplantation.

Abstract 1148Poster Board I-170Graft versus host disease (GVHD) remains a significant cause of morbidity and mortality after allogeneic hematopoietic stem cell transplantation (HCT). In vivo quantitative T-cell depletion using CAMPATH-1h (anti-CD52) has been explored in an effort to prevent acute GVHD. More recently, a regimen consisting of total lymphoid irradiation and anti-thymocyte globulin (ATG) has been shown to polarize T cells towards an inhibitory phenotype potentially reducing the associated risk for GVHD. However, these strategies may be associated with impaired post-transplant immune reconstitution, increased risk of tumor relapse and opportunistic infection. In this study we examined the pattern of cellular immune recovery following T cell depletion with CAMPATH-1h and compared results with an initial cohort of patients undergoing reduced intensity conditioning with TLI and ATG. Immunologic analyses were performed on twenty patients undergoing reduced intensity conditioning in conjunction with low dose CAMPATH -1h (50 mg) and an initial cohort of 5 patients treated with TLI/ATG. Conditioning with CAMPATH-1h resulted in the significant depletion of CD3, CD4, and CD8 T cells in the early post-transplant period and persistence of CD4 T cell depletion (< 200 cells /uL) for more than 6 months. Following TLI/ATG, persistent depletion of CD4+ T cells was also observed but no significant decrease in CD8 T cells was seen. A two-fold increase in circulating CD56+ NK cells, 21.8 to 41.6% (p=0.004), was seen following TLI-ATG, which was not noted following Campath conditioning. CAMPATH-1h conditioning was associated with a significant decrease in mean CD45RO+ memory T cells in the early post-transplant period (27.2 to 5.7% of the total population of nonadherent peripheral blood mononuclear cells, p=0.034). Relative percentages of naïve T cells (CD45RA+), central memory (CD45RO+CD62L+CCR7+) (CM), and effector memory (CD45RO+CD62L-CCR7-) (EM) T cells remained stable in the pre- and post-transplantation period. The CM:EM was 0.6 pre-transplant and at day 60, respectively. In contrast, T cell recovery in early post-transplant following the TLI/ATG regimen was associated with no reduction in CD45RO+ memory T cells. A significant rise in the relative percentages of naïve T cells from 39 to 61.3% (p=0.04), CM cells from 12 to 32.8% (p=0.05), a corresponding fall in EM cells from 57.9 to 32.5% (p=0.10), and a significant change in the CM:EM levels (0.2 pre-transplant, 1.0 day 60 post-transplant) was noted after TLI/ATG. The mean percentage of regulatory T cells as defined by the percentage of CD4+/CD25+ cells that express FoxP3 rose in the early post-transplant period following both regimens (8 to 20.7% at Day 30, p=0.003 in the CAMPATH group; 5.6 to 16.9% at Day 30, p=0.03 in the ATG/TLI group). Functional analyses demonstrated that the T cell proliferative response to the mitogen, Phytohemagglutinin (PHA), was profoundly depressed following CAMPATH-1h with mean SI decreasing from 34 pre-transplant to 1.4 at Day 30. In contrast, treatment with TLI/ATG resulted in no significant change in T cell proliferation in response to PHA with SI only decreasing from 45 pre-transplant to 36 at Day 30. Assessment of T cell polarization following stimulation with PHA or phorbol-ester (PMA)/ionomycin, recipient derived dendritic cells (DCs) or third party DCs demonstrated a rise of CD8+ T cells expressing, IL-4 and IL-10 consistent with a suppressor phenotype. Minimal T cell proliferation was observed following stimulation with patient derived DCs, which is consistent with suppression of the expansion of alloreactive T cells. In summary, both CAMPATH and TLI/ATG result in CD4+ T cell depletion but TLI/ATG resulted in relative preservation of CD8+ T cells, persistence of memory cells, relative preservation of central memory as compared to memory effector cells and intact response to mitogens. TLI/ATG therapy was also associated with the polarization of CD8+ T cells towards a Tc2 phenotype and lack of proliferation in response to recipient derived DCs. As such, TLI/ATG appears to be associated with more modest level of functional T cell depletion characterized by Tc2 polarization and suppression of host/donor alloreactivity. DisclosuresSpitzer:Genzyme: Consultancy. Avigan:Genzyme: Consultancy.

Titanium uptake, induction of RANK‐L expression, and enhanced proliferation of human T‐lymphocytes

There is increasing evidence that titanium ions are released from orthopedic implants by biocorrosion. The aim of this study was to investigate titanium uptake by human T-lymphocytes and its effects on phenotype and proliferation. Freshly isolated human nonadherent peripheral blood mononuclear cells (NA-PBMC), were exposed to TiCl4 [Ti(IV)]. Bioavailability and distribution of Ti(IV) in T-lymphocytes was determined by energy-filtered electron microscopy (EFTEM). The effects of Ti(IV) challenge on nonactivated and PHA-activated cells were assessed by flow cytometric analysis of surface markers, RANK-L production, and proliferation assays. EFTEM colocalized Ti(IV) with phosphorus in the nucleus, ribosomes, cytoplasmic membranes, and the surface membrane of T-lymphocytes. Ti(IV) increased significantly the expression of CD69, CCR4, and RANK-L in a concentration-dependent manner. Titanium enters T-lymphocytes through a currently unknown mechanism and binds to phosphorus-rich cell structures. Titanium influences phenotype and function of T-lymphocytes, resulting in activation of a CD69+ and CCR4+ T-lymphocyte population and secretion of RANK-L. These results strongly suggest the involvement of titanium ions challenged T-lymphocytes in the complex pathophysiological mechanisms of aseptic loosening of orthopedic implants.

Peptide specific expansion of CD8+ T cells by recombinant plate bound MHC/peptide complexes

Development of methods for efficient in vitro stimulation and expansion of peptide specific CD8+ T cells is compelling not only with respect to adoptive T cell therapy but also regarding analysis of T cell responses and search for new immunogenic peptides. In the present study, a new approach to in vitro T cell stimulation was investigated. By use of an antigenic peptide derived from the cytomegalovirus (CMVp) we tested the stimulatory efficacy of recombinant plate bound MHC molecules (PB-MHC), being immobilized in culture plates. A single stimulation of non-adherent peripheral blood mononuclear cells (NA-PBMCs) with PB-MHC/CMVp resulted in significant expansion of CMVp specific CD8+ T cells, which was comparable to that achieved by CMVp pulsed mature dendritic cells (DCs). By repeated exposure of NA-PBMCs to PB-MHC/CMVp more than 60% CMVp specific CD8+ T cells, representing a 240-fold expansion, were reached after only two stimulations. Although stimulation with PB-MHC/CMVp clearly demonstrated efficient peptide specific expansion of CD8+ T cells, there was a tendency to proliferative exhaustion of the cells after 3–4 stimulations. Thus, it will be of interest to examine the effect of new stimulatory cocktails, e.g. cytokines and co-stimulatory molecules, by use of the present rapid and easy-to-use method of expanding peptide specific T cells.

Assessing Identity, Phenotype, and Fate of Endothelial Progenitor Cells

From the paradigm shifting observations of Harvey, Malpighi, and van Leeuwenhoek, blood vessels have become recognized as distinct and dynamic tissue entities that merge with the heart to form a closed circulatory system.1 Vessel structures are comprised predominantly of a luminal layer of endothelial cells that is surrounded by some form of basement membrane, and mural cells (pericytes or vascular smooth muscle cells) that make up the vessel wall. In larger more complex vessel structures the vessel wall is composed of a complex interwoven matrix with nerve components. Understanding the cellular and molecular basis for the formation, remodeling, repair, and regeneration of the vasculature have been and continue to be popular areas for investigation. The endothelium has become a particularly scrutinized cell population with the recognition that these cells may play important roles in maintaining vascular homeostasis and in the pathogenesis of a variety of diseases.2 Although it has been known for several decades that some shed or extruded endothelial cells enter the circulation as apparent contaminants in the human blood stream,3 only more recent technologies have permitted the identification of not only senescent sloughed endothelial cells,4 but also endothelial progenitor cells (EPCs), which have been purported to represent a normal component of the formed elements of circulating blood5 and play roles in disease pathogenesis.6–9 Most citations refer to an article published in 1997 in which Asahara and colleagues isolated, characterized, and examined the in vivo function of putative EPCs from human peripheral blood as a major impetus for generating interest in the field.10 This seminal article presented some evidence to consider emergence of a new paradigm for the process of neovascularization in the form of postnatal vasculogenesis. Since publication of that article, interest in circulating endothelial cells, and particularly EPCs, has soared, …

Natural killer cell-mediated lysis of T cell lines chronically infected with HIV-1

The susceptibility of HIV-1-infected CD4+ T cell lines to natural killer (NK) cell-mediated lysis was examined. Non-adherent peripheral blood mononuclear cells (PBMC) of healthy adults lysed HUT cells chronically infected with the IIIB or WMJ1 strains of HIV-1 to a significantly greater extent than uninfected HUT cells. In contrast, Sup-T1 cells chronically infected with these two strains of HIV-1 were not lysed to a greater extent than uninfected Sup-T1 cells. Clone A1.25-infected Sup-T1 (A1.25/Sup-T1), derived from IIIB-infected Sup-T1 cells (IIIB/Sup-T1), were susceptible to non-adherent PBMC-mediated lysis, as were A1.25-infected HUT cells (A1.25/HUT). When non-adherent PBMC were depleted of CD16 (Leu-11b)+ NK cells by treatment with anti-Leu-11b plus C, lysis of HIV-1-infected HUT or Sup-T1 cells was reduced to low levels, indicating that the lysis was mediated by NK cells. Expression of HIV antigens on these target cells did not correlate with their susceptibility to NK cell-mediated lysis. Depletion of interferon-alpha (IFN-alpha) producing HLA-DR+ cells from non-adherent PBMC had no effect on the magnitude of NK cell-mediated lysis of IIIB or WMJ1-infected HUT cells. In contrast, lysis of A1.25/Sup-T1 or A1.25/HUT cells required the presence of HLA-DR+ cells. IFN-alpha production appeared to be required for NK cell-mediated lysis of A1.25/Sup-T1 or A1.25/HUT cells, while lysis of HUT cells infected with the WMJ1 or IIIB strains of HIV-1 was IFN-alpha independent. These results indicate considerable variability in the susceptibility of different HIV-1 infected T cell lines to NK cell-mediated lysis and suggest the existence of alternative mechanisms of activation of NK cells for lysis of HIV-1-infected T cell lines.

Systemic and Brain Macrophage Infections in Relation to the Development of Simian Immunodeficiency Virus Encephalitis

The brains of individuals with lentiviral-associated encephalitis contain an abundance of infected and activated macrophages. It has been hypothesized that encephalitis develops when increased numbers of infected monocytes traffic into the central nervous system (CNS) during the end stages of immunosuppression. The relationships between the infection of brain and systemic macrophages and circulating monocytes and the development of lentiviral encephalitis are unknown. We longitudinally examined the extent of monocyte/macrophage infection in blood and lymph nodes of pigtailed macaques that did or did not develop simian immunodeficiency virus encephalitis (SIVE). Compared to levels in macaques that did not develop SIVE, more ex vivo virus production was detected from monocyte-derived macrophages and nonadherent peripheral blood mononuclear cells (PBMCs) from macaques that did develop SIVE. Prior to death, there was an increase in the number of circulating PBMCs following a rise in cerebrospinal fluid viral load in macaques that did develop SIVE but not in nonencephalitic macaques. At necropsy, macaques with SIVE had more infected macrophages in peripheral organs, with the exception of lymph nodes. T cells and NK cells with cytotoxic potential were more abundant in brains with encephalitis; however, T-cell and NK-cell infiltration in SIVE and human immunodeficiency virus encephalitis was more modest than that observed in classical acute herpes simplex virus encephalitis. These findings support the hypothesis that inherent differences in host systemic and CNS monocyte/macrophage viral production are associated with the development of encephalitis.

Peripheral blood mononuclear cells increase the permeability of dengue virus-infected endothelial cells in association with downregulation of vascular endothelial cadherin

Plasma leakage is one of the characteristic features of dengue haemorrhagic fever. The interaction among peripheral blood mononuclear cells (PBMCs), dengue virus and endothelial cells was analysed in vitro. Human umbilical vein endothelial cells (HUVECs) were infected with dengue-2 virus (DV-2) at an m.o.i. of 0.5 p.f.u. per cell. PBMCs were added to DV-2-infected HUVECs, and transendothelial electrical resistance (TEER) and transalbumin permeability were assessed. Dengue virus infection at an m.o.i. of 0.5 p.f.u. per cell alone did not decrease the TEER, but addition of PBMCs decreased the TEER, increased the albumin permeability and induced morphological changes of HUVECs. The extent of the decrease was more profound with adherent PBMCs than with non-adherent PBMCs. The expression of vascular endothelial cadherin (VE-cadherin) was examined using real-time RT-PCR and immunofluorescence. Addition of PBMCs to DV-2-infected HUVECs decreased the levels of mRNA transcripts and cell-surface expression of VE-cadherin. The results indicate that PBMCs increased the permeability of DV-2-infected HUVECs and that the increased permeability was concomitant with morphological change and the decrease in VE-cadherin expression. The results suggest that functional impairment of the DV-2-infected HUVEC monolayer was caused by interaction with PBMCs.

Vaccination strategy to target lysyl oxidase-like 4 in dendritic cell based immunotherapy for head and neck cancer

Overexpression of lysyl oxidase (LOX) is associated with the invasive potential of metastatic breast and head and neck cancer (HNC) cells and reduced metastasis-free and overall survival. Recently, we have demonstrated up-regulation of a new member of the LOX family, lysyl oxidase-like 4 (LOXL4), in invasive HNC revealed a significant correlation between LOXL4 expression and local lymph node metastases and higher tumour stages. The objective of this study was to examine whether cellular LOXL4 may provide an effective target for cell-meditated immunotherapy in invasive tumours associated with LOXL4 overexpression. As a feasibility study we expressed LOXL4 mRNA in immature dendritic cells derived from human peripheral blood mononuclear cells (PBMC). LOXL4 protein expression was ascertained using Western blotting and immunocytochemistry with polyclonal rabbit anti-LOXL4 antibody. The successfully transfected immature dendritic cells (DCs) were induced to mature with GM-CSF, IL-4, IL-1beta, TNF-alpha, IL-6, and PGE2, and then used to stimulate T cell enriched non-adherent fraction of PBMC. LOXL4 specific T cell stimulation induced cytotoxic T lymphocyte (CTL) response was monitored using IFN-gamma secretion from the non-adherent PBMC fraction exposed to mature, LOXL4 transfected DCs acting as the antigen presenting target cells. LOXL4-DC stimulated T cells produced higher IFN-gamma secretion compared to unstimulated T cells and T cells stimulated with untransfected DCs, in the presence of the pan-DR-epitope (PADRE). These initial results demonstrated the potential for LOXL4-transfected DCs to serve as efficient tumour vaccine and support their suitability as a vaccination strategy applicable to cancer patients with tumour specific up-regulation of LOXL4.

Engulfment of apoptotic cells expressing HCV proteins leads to differential chemokine expression and STAT signaling in human dendritic cells

In the majority of cases, infection with hepatitis C virus (HCV) becomes chronic and is often associated with impaired innate and adaptive immune responses. The mechanisms underlying viral persistence and lack of protective immunity are poorly understood. Considering that dendritic cells (DCs) play critical roles in initiating and modulating immune responses, we explored the effect of HCV proteins on DC gene and protein expression, phenotype, and function. Human DCs were generated following plastic adherence of monocytes and culture with granulocyte-macrophage colony-stimulating factor and interleukin-4 (IL-4) from normal subjects. Autologous nonadherent peripheral blood mononuclear cells were infected with vaccinia constructs expressing various HCV proteins (core-E1, NS5A, NS5B) or an irrelevant protein beta-galactosidase (beta-gal) as the control, induced to undergo apoptosis, then co-cultured with DCs. Between 2% and 10% of the genes probed in a cDNA nylon array were differentially regulated within DCs that had engulfed HCV proteins. In particular, the presence of intracellular NS5A led to increased transcriptional and protein expression of IL-8 (CXCL-8), a chemokine with proinflammatory and anti-interferon properties, and impaired interferon induction of signal transducers and activators of transcription 1 (STAT1) serine and tyrosine and STAT2 tyrosine phosphorylation. These data provide novel mechanisms by which HCV subverts antiviral host immunity.

Molecular Markers of Eosinophilopoiesis: Multiplex Q-PCR Analysis of GATA-1, MBP and IL-5 Receptor mRNA Expression in Peripheral Blood

RATIONALE: Using colony assays and flow cytometry, we have shown that eosinophil/basophil (Eo/B) progenitor phenotype and function are associated with atopic risk at birth and early childhood clinical outcomes. We have also recently demonstrated that real-time polymerase chain reaction (Q-PCR) can reveal kinetic patterns of expression in cord blood (CB) of several Eo/B lineage-specific genes, specifically GATA-1, MBP and IL-5Rα, as surrogate molecular markers of Eo/B differentiation. These same methods have yet to be established in peripheral blood (PB) samples. Our objective was to determine the kinetic patterns of expression of CB Eo/B-lineage specific genes in PB. METHODS: PB non-adherent mononuclear cells (PB NAMNC) were isolated from random fresh samples, and incubated in the presence of IL-5. At 24, 48, and 72h post-stimulation, RNA was isolated, reverse transcribed, and expression of IL-5Rα, GATA-1, and MBP was determined utilizing multiplex Q-PCR. Relative expression ratios of stimulated to un-stimulated cells were calculated using the delta-delta Ct method. RESULTS: Stimulation of PB NANMC with IL-5 resulted in an up-regulation of GATA-1 expression, peaking at 24h, with a slower return to baseline expression than that observed in CB. MBP expression was minimally altered at all time points, compared to CB, where slow up-regulation, maximal at 72h, had been observed. There was completely stable expression IL-5Rα, similar to that seen in CB. CONCLUSIONS: Multiplex Q-PCR analysis of mRNA from PB demonstrates expression of critical Eo/B lineage-specific events. Further investigation of the validity and utility of Q-PCR analyses of PB for surrogate, molecular markers Eo/B differentiation is underway.

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Meeting program

A primary study of immunotherapy with carcinoembryonic antigen peptide-pulsed, autologous human cultured dendritic cells in patients with advanced non-small cell lung cancer

Dendritic cell (DC)-based immunotherapy is a new approach and effective for some malignant tumors. The aim of this study is to observe the efficacy and toxicity of immunotherapy with carcinoembryonic antigen (CEA) peptide-pulsed DCs in patients with refractory advanced lung cancer. Lung cancer patients with high CEA expression were enrolled into this project. Autologous DCs were generated from patients' plastic-adherent peripheral blood mononuclear cells and loaded with CEA 5 days later. Cytokine-induced killer cells (CIK) were cultured from non-adherent peripheral blood mononuclear cells. DCs and CIK were transfused to patients. Responses and toxicities were observed. A total of 22 patients with lung cancer received DCs immunotherapy. DCs doses were 2.5×10⁶-9.6×10⁷ (5.03×10⁶). CIK doses were 3.4×10⁸-46×10⁸. CD3, CD8, NK and IFN-γ levels obviously increased after treatment (P < 0.05). The 1-year survival rate was 68.2% (15/22). Main toxicities were fever and rash. DCs-based immunotherapy is feasible and safe to patients with lung cancer.

Inhibition of natural killer (NK) cell activity against varicella-zoster virus (VZV)-infected fibroblasts and lymphocyte activation in response to VZV antigen by nitric oxide-releasing agents.

The addition of nitric oxide (NO)-releasing agents, S-nitroso-N-acetyl-DL-penicillamine (SNAP), 1-hydroxy-2-oxo-2,3-bis(2-aminoethyl)-1-triazene (NOC18), 30{(+/-)-(E)-ethyl-2'-[(E)-hydroxyimino]-5-nitro-3-hexenecarbam oyl} -pyridine (NOR4) significantly inhibited NK cell activity against VZV-infected cells, while antibody-dependent cell-mediated cytotoxicity (ADCC) against VZV-infected cells was unaffected. Interferon-alpha (IFN-alpha) production by non-adherent peripheral blood mononuclear cells (NPBMC) cultured with VZV-infected cells was decreased by the addition of NO-releasing agents. Lymphocyte proliferation and the expression IL-2 receptor (CD25) in response to VZV antigen were also inhibited by the addition of NO-releasing agents. These results suggest that the production of NO by an inflammatory process may lead to inhibition of NK cell- and T cell-mediated immunity to VZV infection.