Effectual Labeling of Natural Killer Cells with Upconverting Nanoparticles by Electroporation for In Vivo Tracking and Biodistribution Assessment.

Natural killer (NK) cells, which are cytotoxic lymphocytes of the innate immune system and recognize cancer cells via various immune receptors, are promising agents in cell immunotherapy. To utilize NK cells as a therapeutic agent, their biodistribution and pharmacokinetics need to be evaluated following systemic administration. Therefore, in vivo imaging and tracking with efficient labeling and quantitative analysis of NK cells are required. However, the lack of the phagocytic capacity of NK cells makes it difficult to establish breakthroughs in cell labeling and subsequent in vivo studies. Herein, an effective labeling of upconverting nanoparticles (UCNPs) in NK cells is proposed using electroporation with high sensitivity and stability. The labeling performance of UCNPs functionalized with carboxy-polyethylene glycol (PEG) is better than with methoxy-PEG or with amine-PEG. The labeling efficiency becomes higher, but cell damage is greater as electric field increases; thus, there is an optimum electroporation condition for internalization of UCNPs into NK cells. The tracking and biodistribution imaging analyses of intravenously injected NK cells show that the labeled NK cells are initially distributed primarily in lungs and then spread to the liver and spleen. These advances will accelerate the application of NK cells as key components of immunotherapy against cancer.

A novel CD2 staining-based flow cytometric assay for assessment of natural killer cell cytotoxicity.

BACKGROUND: Assessing cytotoxicity is fundamental to studying natural killer (NK) cell function. Various radioactive and non-radioactive cytotoxicity assays measuring target cell death have been developed. Among these methods, the most commonly used 51 Chromium-release assay (CRA) and flow cytometry-based cytotoxicity assays (FCCs) are the major representatives. Nonetheless, several drawbacks, including dye leakage and the potential effects of prior labeling on cells, curb the broad applicability of the FCCs. METHODS: Here, we report a rapid FCC for quantifying target cell death after co-incubation with NK cells. In this assay, after 4 hours of NK cell-target cell co-incubation, fluorochrome-conjugated CD2 antibody was used to identify NK cells, and SYTOX Green and Annexin V-FITC were further used to detect target cell death in CD2-negative population. In parallel, both CRA and FCC assay using CFSE/ 7-AAD were performed to validate the reproducibility and replicability. RESULTS: We observed that CD2 is exclusively positive on NK cells other than the most common hematological target tumor cells, such as K562, HL60, MOLM13, Raji, NCI-H929, rpmi8226, MM.1S, and KMS11. Assessment of target cell death using the CD2-based FCC shows a significantly higher percent specific lysis of the target cells compared to the standard CRA and the FCC assay using CFSE and 7-AAD. CONCLUSIONS: We demonstrated that this CD2-based FCC is a fast, simple, and reliable method for evaluating NK cell cytotoxicity.

Assessment of Human Natural Killer Cell Events Driven by FcγRIIIa Engagement in the Presence of Therapeutic Antibodies.

One mechanism of action for clinical efficacy by therapeutic antibodies is the promotion of immune-related functions, such as cytokine secretion and cytotoxicity, driven by FcγRIIIa (CD16) expressed on natural killer (NK) cells. These observations have led to research focusing on methods to increase Fc receptor-mediated events, which include removal of a fucose moiety found on the Fc portion of the antibody. Further studies have elucidated the mechanistic changes in signaling, cellular processes, and cytotoxic characteristics that increase ADCC activity with afucosylated antibodies. Additionally, other studies have shown the potential benefits of these antibodies in combination with small molecule inhibitors. These experiments demonstrated the molecular and cellular mechanisms underlying the benefits of using afucosylated antibodies in combination settings. Many of these observations were based on an artificial in vitro activation assay in which the FcγRIIIa on human NK cells was activated by therapeutic antibodies. This assay provided the flexibility to study downstream effector NK cell functions, such as cytokine production and degranulation. In addition, this assay has been used to interrogate signaling pathways and identify molecules that can be modulated or used as biomarkers. Finally, other therapeutic molecules (i.e., small molecule inhibitors) have been added to the system to provide insights into the combination of these therapeutics with therapeutic antibodies, which is essential in the current clinical space. This manuscript aims to provide a technical foundation for performing this artificial human NK cell activation assay. The protocol demonstrates key steps for cell activation as well as potential downstream applications that range from functional readouts to more mechanistic observations.

Single cell arrays of hematological cancer cells for assessment of lymphocyte cytotoxicity dynamics, serial killing, and extracellular molecules.

Cytotoxicity exerted by cytotoxic lymphocytes against cancer cells is an essential cellular function for successful cancer immunotherapy. Standard cytotoxicity assays mostly provide population level information, whereas live cell imaging-based cytotoxicity assays can assess single cell level heterogeneity. However, long term tracking of individual cytotoxic lymphocyte-hematological cancer cell interactions is technically challenging because both cells can float around and form multi-cellular aggregates. To overcome this limitation, single hematological cancer cell arrays with immobilized hematological cancer cells are fabricated using microwell arrays. Using this new platform, single cell level natural killer (NK) cell cytotoxicity against leukemic cells is quantitatively assessed. Depending on microwell surface adhesiveness and inter-microwell distances, distinct modes of NK-leukemic cell interactions that result in different NK cell cytotoxicity are observed. For microwell arrays coated with bovine serum albumin, which prevents cell adhesion, NK cells stably contacted cancer cells with rounded morphologies, whereas for microwell arrays coated with fibronectin (FN), which triggers integrin signals, NK cells contacting cancer cells exhibited dynamic behaviors with elongated morphologies and constantly explored extracellular spaces by membrane extension. In addition, FN on extracellular spaces facilitate NK cell detachment from leukemic cells after killing by providing anchorage for force transmission, and promote cytotoxicity and serial killing. Single hematologic cell arrays are not only an efficient method for lymphocyte cytotoxicity analysis but also a useful tool to study the role of signaling molecules in extracellular spaces on lymphocyte cytotoxicity.

Cost-effective and fast KIR gene-content genotyping by multiplex melting curve analysis.

Killer cell immunoglobulin-like receptor (KIR) genes encode cell surface molecules that recognize HLA molecules and modulate the activity of natural killer (NK) cells. KIR genes exhibit presence and absence polymorphism, which generates a variety of gene-content haplotypes in worldwide populations. KIR gene-content variation is implicated in many diseases and is also important for placentation and transplantation. Because of the complexity of KIR polymorphism, variation in this family is still mostly studied at the gene-content level, even with the advent of next-generation sequencing (NGS) methods. Gene-content determination is generally expensive and/or time-consuming. To overcome these difficulties, we developed a method based on multiplex polymerase chain reaction with specific sequence primers (PCR-SSP) followed by melting curve analysis that allows cost-effective, precise and fast generation of results. Our method was 100% concordant with a gel-based method and 99.9% concordant with presence and absence determination by NGS. The limit of detection for accurate typing was 30 ng of DNA (0.42 µM) with 260/230 and 260/280 ratios as low as 0.19 and of 0.44. In addition, we developed a user-friendly Java-based computational application called killerPeak that interprets the raw data generated by Viia7 or QuantStudio 7 quantitative PCR machines and reliably exports the final genotyping results in spreadsheet file format. The combination of a reliable method that requires low amount of DNA with an automated interpretation of results allows scaling the KIR genotyping in large cohorts with reduced turnaround time.

Flow cytometric assessment of leukocyte kinetics for the monitoring of tissue damage.

Leukocyte populations quickly respond to tissue damage, but most leukocyte kinetic studies are not based on multiparameter flow cytometry. We systematically investigated several blood leukocyte populations after controlled tissue damage. 48 patients were assigned to either an anterior or posterolateral total hip arthroplasty. Peripheral blood was collected pre-operatively and at 2 h, 24 h, 48 h, 2 and 6 weeks postoperatively and assessed by flow cytometry for absolute counts of multiple leukocyte populations using standardized EuroFlow protocols. Absolute counts of leukocyte subsets differed significantly between consecutive time points. Neutrophils increased instantly after surgery, while most leukocyte subsets initially decreased, followed by increasing cell counts until 48 h. At two weeks all leukocyte counts were restored to pre-operative counts. Immune cell kinetics upon acute tissue damage exhibit reproducible patterns, which differ between the leukocyte subsets and with "opposite kinetics" among monocyte subsets. Flow cytometric leukocyte monitoring can be used to minimally invasively monitor tissue damage.

Assessment of new HDAC inhibitors for immunotherapy of malignant pleural mesothelioma.

Background: Malignant pleural mesothelioma (MPM) is a very rare and highly aggressive cancer of the pleura associated in most cases with asbestos exposure. To date, no really efficient treatments are available for this pathology. Recently, it has been shown that epigenetic drugs, particularly DNA methylation or histone acetylation modulating agents, could be very efficient in terms of cytotoxicity for several types of cancer cells. We previously showed that a hypomethylating agent (decitabine) and a histone deacetylase inhibitor (HDACi) (valproic acid (VPA)) combination was immunogenic and led to the induction of an anti-tumor immune response in a mice model of mesothelioma. However, VPA is not very specific, is active at millimolar concentrations and is responsible for side effects in clinic. To improve this approach, we studied four newly synthetized HDACi, two hydroxamates (ODH and NODH) and two benzamides (ODB and NODB), in comparison with VPA and SAHA. We evaluated their toxicity on immune cells and their immunogenicity on MPM cells in combination with decitabine. Results: All the tested HDACi were toxic for immune cells at high concentrations. Combination with decitabine increased toxicity of HDACi only towards T-cell clone. A decrease in the proportion of regulatory T cells and natural killer cells was observed in particular with VPA and ODH. In MPM cells, all HDACi combinations induced NY-ESO-1 cancer testis antigen (CTA) expression and the recognition of the treated cells by a NY-ESO-1 specific T-CD8 clone. However, for MAGE-A1, MAGE-A3 and XAGE-1b mRNA expression, the results obtained depended on the HDACi used and on the CTA studied. Depending on the MPM cell line studied, molecules alone increased moderately PD-L1 expression. When combined, a higher stimulation of this immune check point inhibitor expression was observed. Decitabine-induced anti-viral response seemed to be inhibited in the presence of HDACi. Conclusions: This work shows that the combination of decitabine and HDACi could be of interest for MPM immunotherapy. However, this combination induced PD-L1 expression which suggests that an association with anti-PD-L1 therapy should be performed to induce an efficient anti-tumor immune response.

Assessment of interactions of efavirenz solid drug nanoparticles with human immunological and haematological systems.

BACKGROUND: Recent work has developed solid drug nanoparticles (SDNs) of efavirenz that have been demonstrated, preclinically, improved oral bioavailability and the potential to enable up to a 50% dose reduction, and is currently being studied in a healthy volunteer clinical trial. Other SDN formulations are being studied for parenteral administration, either as intramuscular long-acting formulations, or for direct administration intravenously. The interaction of nanoparticles with the immunological and haematological systems can be a major barrier to successful translation but has been understudied for SDN formulations. Here we have conducted a preclinical evaluation of efavirenz SDN to assess their potential interaction with these systems. Platelet aggregation and activation, plasma coagulation, haemolysis, complement activation, T cell functionality and phenotype, monocyte derived macrophage functionality, and NK cell function were assessed in primary healthy volunteer samples treated with either aqueous efavirenz or efavirenz SDN. RESULTS: Efavirenz SDNs were shown not to interfere with any of the systems studied in terms of immunostimulation nor immunosuppression. Although efavirenz aqueous solution was shown to cause significant haemolysis ex vivo, efavirenz SDNs did not. No other interaction with haematological systems was observed. Efavirenz SDNs have been demonstrated to be immunologically and haematologically inert in the utilised assays. CONCLUSIONS: Taken collectively, along with the recent observation that lopinavir SDN formulations did not impact immunological responses, these data indicate that this type of nanoformulation does not elicit immunological consequences seen with other types of nanomaterial. The methodologies presented here provide a framework for pre-emptive preclinical characterisation of nanoparticle safety.

Natural Killer Cell Assessment in Peripheral Circulation and Bronchoalveolar Lavage Fluid of Patients with Severe Sepsis: A Case Control Study.

Sepsis is a complex systemic inflammatory syndrome, the most common cause of which is attributed to systemic underlying bacterial infection. The complete mechanisms of the dynamic pro- and anti-inflammatory processes underlying the pathophysiology of sepsis remain poorly understood. Natural killer (NK) cells play a crucial role in the pathophysiology of sepsis, leading to exaggerated inflammation due their rapid response and production of pro-inflammatory cytokines such as interferon gamma (IFN-γ). Several studies have already shown that NK cells undergo lymphopenia in the peripheral blood of patients with sepsis. However, our understanding of the mechanisms behind its cellular trafficking and its role in disease development is restricted to studies in animal models. In this study, we aimed to compare the human NK cell subset (CD56bright or dim) levels in the peripheral blood and bronchoalveolar lavage (BAL) fluid of sepsis patients. We conducted a case-control study with a sample size consisting of 10 control patients and 23 sepsis patients enrolled at the Hospital Cajuru (Curitiba/PR, Brazil) from 2013 to 2015. Although we were able to confirm previous observations of peripheral blood lymphopenia, no significant differences were detected in NK cell levels in the BAL fluid of these patients. Overall, these findings strengthened the evidence that peripheral blood lymphopenia is likely to be associated with cell death as a consequence of sepsis.

Assessment of NK Cell Metabolism.

There has been increasing recognition of the importance of metabolism on immune cell differentiation, homeostasis, and function. Recently, our lab and others have begun to investigate the metabolic requirements for NK cell differentiation and activation. Here, we describe approaches for the in vitro assessment of NK cell metabolism. We present methods for using inhibitors to alter cellular metabolism, measurement of intracellular ATP in NK cells, assessment of real-time glycolysis and oxidative phosphorylation by an extracellular flux assay from Seahorse Biosciences, and some basic protocols for stimulation of NK cells via cytokines and receptors.

In Vitro Assessment of Human Natural Killer Cell Migration and Invasion.

Cell invasion assays are important to obtain valuable functional data relating to tissue migration and invasion of effector lymphocytes. Boyden chamber invasion assays represent a reductionist system that allows for easy manipulation using various extracellular matrix (ECM) components addressing migratory functions or invasion into/through a three-dimensional matrix where migration and invasion inhibitors as well as stimulators can be added. Presented here is a description using the Transwell(®) system where invasion and migration can be studied. It constitutes an inner cell culture well with a (PET) polycarbonate filter with 3-8 µm pores that allow for cells to transverse to the bottom chamber where they can be recorded by various methods (Fig. 1). The polycarbonate filters may be coated with ECM proteins for migration and adhesive studies or covered with a thick layer that occludes the pores to address matrix degradation, i.e., invasion as described in this chapter.

Assessment of Natural Killer Cell Cytotoxicity Using Image Cytometry Method.

Although natural killer (NK) cells produce various cytokines that regulate other lymphocytes of the immune system, the primary effector function of NK cells is the direct cytolysis of their targets. Hence analyzing the cytotoxic potential of these lymphocytes is fundamental to understanding their biology and their clinical impact. We have previously shown that release-based cytotoxicity assays, such as calcein release assay, could potentially underestimate percent specific lysis if the entrapped reporter is not completely released and demonstrated that an Image cytometry method can overcome this caveat. In this chapter, we describe a detailed methodology to quantitate NK cell cytotoxicity using the Cellometer Vision Image Cytometry system.

Isolation and (111)In-Oxine Labeling of Murine NK Cells for Assessment of Cell Trafficking in Orthotopic Lung Tumor Model.

A noninvasive in vivo imaging method for NK cell trafficking is essential to gain further understanding of the pathogenesis of NK cell mediated immune response to the novel cancer treatment strategies, and to discover the homing sites and physiological distribution of NK cells. Although human NK cells can be labeled for in vivo imaging, little is known about the murine NK cell labeling and its application in animal models. This study describes the isolation and ex vivo radiolabeling of murine NK cells for the evaluation of cell trafficking in an orthotopic model of human lung cancer in mice. Scid-Tg(FCGR3A)Blt transgenic SCID mice were used to isolate NK cells from mouse splenocytes using the CD49b (DX5) MicroBeads positive selection method. The purity and viability of the isolated NK cells were confirmed by FACS analysis. Different labeling buffers and incubation times were evaluated to optimize (111)In-oxine labeling conditions. Functionality of the radiolabeled NK cell was assessed by (51)Cr-release assay. We evaluated physiological distribution of (111)In-oxine labeled murine NK cells in normal SCID mice and biodistribution in irradiated and nonirradiated SCID mice with orthotopic A549 human lung tumor lesions. Imaging findings were confirmed by histology. Results showed that incubation with 0.011 MBq of (111)In-oxine per million murine NK cells in PBS (pH 7.4) for 20 min is the best condition that provides optimum labeling efficiency without affecting cell viability and functionality. Physiological distribution in normal SCID mice demonstrated NK cells homing mainly in the spleen, while (111)In released from NK cells was excreted via kidneys into urine. Biodistribution studies demonstrated a higher lung uptake in orthotopic lung tumor-bearing mice than control mice. In irradiated mice, lung tumor uptake of radiolabeled murine NK cells decreased between 24 h and 72 h postinjection (p.i.), which was accompanied by tumor regression, while in nonirradiated mice, radiolabeled NK cells were retained in the lung tumor lesions up to 72 h p.i. without tumor regression. In tumor-bearing mice that were only irradiated but did not receive radiolabeled murine NK cells, a high tumor burden was observed at 72 h p.i., which indicates that irradiation in combination with murine NK cell allocation, but not irradiation alone, induced a remarkable antitumor effect in the orthotopic A549 lung tumor bearing mouse model. In conclusion, we describe a method to evaluate murine NK cell trafficking and biodistribution, which can be used to determine potential effects of immune-mediated therapeutic agents on NK cell biodistribution.

Microfluidic-based, live-cell analysis allows assessment of NK-cell migration in response to crosstalk with dendritic cells.

Migration and localization of NK cells into peripheral tissues are tightly regulated under normal and pathological conditions. The physiological importance of NK cell-DC crosstalk has been well documented. However, the ways in which DCs regulate the migratory properties of NK cells (such as chemotaxis, chemokinesis, chemo-repulsion) are not fully defined in vitro. Here, we employed a microfluidic platform to examine, at the single-cell level, C57BL/6 NK-cell migrations in a stable chemical gradient. We observed that soluble factors released by the immature and LPS-activated mature DCs induced a high level of chemotactic movement of IL-2-activated NK cells in vitro. We confirmed these findings in a standard trans-well migration assay, and identified CXCR3 as a key receptor on the NK cells that mediated the migration. More interestingly, we revealed a novel function of granulocyte macrophage colony-stimulating factor in repulsing NK-cell migrations. The future uses of such microfluidic device in the systematic evaluations of NK-cell migratory responses in NK cell-DC crosstalk will provide new insights into the development of DC-based NK-cell therapies against tumor and infections.

Assessment of copy-number variation in the NKG2C receptor gene in a single-tube and characterization of a reference cell panel, using standard polymerase chain reaction.

Natural killer (NK) and T-lymphocytes monitor human leukocyte antigen (HLA)-E expression through CD94:NKG2 heterodimers. Structural polymorphism is not a hallmark for NK-complex genes on chromosome 12, except for complete NKG2C deletion in some humans. We present a method for fast, simple and accurate assessment of NKG2C copy-number variation - presence or absence in the genome of an NKG2C gene, in homo- or heterozygosis, is detected by a single conventional polymerase chain reaction that yields amplicons of different lengths in each genotype. We have also determined the NKG2C genotypes of a reference cell panel comprising 13 NK- and tumour-cell lines and 39 Epstein-Barr virus transformed cells from the International Histocompatibility Workshop. Our results should facilitate research on the importance of NKG2C and its deletion for immunity.

Expansion, purification, and functional assessment of human peripheral blood NK cells.

Natural killer (NK) cells play an important role in immune surveillance against a variety of infectious microorganisms and tumors. Limited availability of NK cells and ability to expand in vitro has restricted development of NK cell immunotherapy. Here we describe a method to efficiently expand vast quantities of functional NK cells ex vivo using K562 cells expressing membrane-bound IL21, as an artificial antigen-presenting cell (aAPC). NK cell adoptive therapies to date have utilized a cell product obtained by steady-state leukapheresis of the donor followed by depletion of T cells or positive selection of NK cells. The product is usually activated in IL-2 overnight and then administered the following day. Because of the low frequency of NK cells in peripheral blood, relatively small numbers of NK cells have been delivered in clinical trials. The inability to propagate NK cells in vitro has been the limiting factor for generating sufficient cell numbers for optimal clinical outcome. Some expansion of NK cells (5-10 fold over 1-2 weeks) has be achieved through high-dose IL-2 alone. Activation of autologous T cells can mediate NK cell expansion, presumably also through release of local cytokine. Support with mesenchymal stroma or artificial antigen presenting cells (aAPCs) can support the expansion of NK cells from both peripheral blood and cord blood. Combined NKp46 and CD2 activation by antibody-coated beads is currently marketed for NK cell expansion (Miltenyi Biotec, Auburn CA), resulting in approximately 100-fold expansion in 21 days. Clinical trials using aAPC-expanded or -activated NK cells are underway, one using leukemic cell line CTV-1 to prime and activate NK cells without significant expansion. A second trial utilizes EBV-LCL for NK cell expansion, achieving a mean 490-fold expansion in 21 days. The third utilizes a K562-based aAPC transduced with 4-1BBL (CD137L) and membrane-bound IL-15 (mIL-15), which achieved a mean NK expansion 277-fold in 21 days. Although, the NK cells expanded using K562-41BBL-mIL15 aAPC are highly cytotoxic in vitro and in vivo compared to unexpanded NK cells, and participate in ADCC, their proliferation is limited by senescence attributed to telomere shortening. More recently a 350-fold expansion of NK cells was reported using K562 expressing MICA, 4-1BBL and IL15. Our method of NK cell expansion described herein produces rapid proliferation of NK cells without senescence achieving a median 21,000-fold expansion in 21 days.

Assessment of immune function after short-term administration of recombinant human growth hormone in healthy young males.

Growth hormone (GH) is a commonly used drug aimed at improving sport performance. The aim of this study is to evaluate the immunomodulatory effects of short-term administration of recombinant GH (rhGH) in healthy young males. NK cell number, activity and phenotype, T cell number, CD4(+) (Th1/Th2) cytokine production of IL2, IL4, IL6, IL10, TNF-α and IFN-γ and CD4(+)/CD8(+) ratio with particular attention to the possible correlation to IGF-I production were investigated. 30 males (27 ± 9 years) were randomly assigned to placebo (n = 15) or drug (rhGH) 1 mg/day groups (n = 15) with daily injection for 7 days. IGF-I plasma concentration and flow cytometry data were generated at baseline and days 8, 15, 22 and 29 post injection. Data analysis used General Linear Model with repeated measures, Bonferroni correction factor and significance at p ≤ 0.05. Serum IGF-I levels (ng/mL) increased significantly (p ≤ 0.01) on day 8 (0.48 ± 0.78) after injections compared to baseline (0.31 ± 0.07) and days 15 (0.33 ± 0.06), 22 (0.29 ± 0.05) and 29 (0.29 ± 0.06). A significant time effect was noted in IL10 secretion (pg/mL) from day 15 (P = 35.14 ± 19.93, rhGH = 26.63 ± 16.39) to days 22 (P = 61.32 ± 20.41, rhGH = 74.99 ± 46.91) and 29 (P = 101.98 ± 67.25, rhGH = 107.74; ± 122.58). There was no correlation between IGF-I and NK activity, phenotype or number along with T lymphocyte number, CD4(+)/CD8(+) ratio or Th1 and Th2 cytokine production. In conclusion, cytokine secretion spectrum was not affected by short-term rhGH administration in young males.

Acquisition, preparation, and functional assessment of human NK cells for adoptive immunotherapy.

Human natural killer (NK) cells, a subset of peripheral blood lymphocytes that lack a T- or B-cell receptor, play a crucial role in the innate immune response to viruses and malignant cells. NK cells differentiate infected or malignant cells from normal cells by a complex balance between activating and inhibitory receptor-ligand interactions. Unlike T cells, NK cells do not proliferate in vitro in response to simple crosslinking of a single activating receptor. While many methods to study T-cell function and phenotype can also be applied to NK cells, this chapter addresses methods that are unique to the preparation and assessment of human NK cells for immunotherapy.

Comprehensive assessment of chemokine expression profiles by flow cytometry.

The chemokines are a large family of mainly secreted molecules involved in the regulation of numerous physiological and pathophysiological processes. Despite many years of investigation, the precise cellular sources of most chemokines have remained incompletely defined as a consequence of the limited availability of suitable reagents to visualize the expression of chemokine proteins at the single-cell level. Here, we developed a simple flow cytometry-based assay using commercially available chemokine-specific antibodies for efficient cell-associated detection of 37 of 39 murine chemokines. To demonstrate the utility of this methodology, we used it to reevaluate the nature of homeostatic chemokines in the hematopoietic compartment, to delineate the complete chemokine profiles of NK cells and B cells in response to major polyclonal stimuli, and to assess the chemokine response of DCs to bacterial infection. The versatility of this analytical methodology was further demonstrated by its application to selected human chemokines and should greatly facilitate any future investigation into chemokine biology at large.

Assessment of NK cells response to hepatocyte derived chemotactic agents.

This project was aimed to examine the NK92 cells response as the CXC chemokine responder cells in rat model of liver disorder and injuries. Hepatocytes were isolated from Sprague-dawly rats and cultured on collagen type 1. Migration of NK92 cells was assessed using a 48 well micro-chemotaxis technique. Transwell chambers were positioned faced up, blocked Medium supernatant (500 microL) obtained from hepatocytes cultures were placed into the lower compartment of each Transwell. The upper compartment was filled with either 500 microL of NK92 cells. After washing, Membrane-attached cells were fixed; stained and Membrane-attached cells were counted by light microscopy and/or by size gating (9-14 microm) with an automated counter system. Human NK92 cells were attracted to recombinant human IP-10 in a concentration and time-dependent manner. NK92 cells also exhibited a chemotactic response to medium harvested from primary hepatocyte cultures. Isolated and cultured hepatocytes express several different chemokines. Although we identified that medium from hepatocyte cultures contains specific chemokines by immunoblotting, there is potential that migration assays detected yet other chemokines and other factors such as complement components. In this report, we demonstrated that hepatocytes expressed factors that were chemoattractive for human NK92 cells and that the factors must interact with the repertoire of receptors responsible for recruitment of these cells.