Supernatant levels of IFN-, TNF-, GM-CSF, IL-8, MIP-1, and RANTES were determined by multiplex assay using the Luminex system (Luminex Co


Supernatant levels of IFN-, TNF-, GM-CSF, IL-8, MIP-1, and RANTES were determined by multiplex assay using the Luminex system (Luminex Co.) and human-specific bead sets (R&D systems; sensitivity 0.5C3.0 pg/mL) and final pg/mL values adjusted for background for each condition were determined using the following equation: [NK cells + Raji cells pg/mL] ? [(NK cells alone pg/mL) + (Raji cells alone pg/mL)]. 51-Chromium Release Cytotoxicity Assay Direct cytotoxicity assays were performed by standard 4-hour 51Cr-release assays using effector cells pre-treated with reagents and Raji cells as targets. after 24 and 48 hours of culture in human serum. NK cell production of IFN-, TNF-, GM-CSF, IL-8, MIP-1 and RANTES was differentially induced in the presence of recombinant reagents and Raji targets. Moreover, significant increases in NK cell degranulation and enhancement of IFN- production against primary ALL and CLL targets were induced with reagent treatment of resting NK cells. In conclusion, BiKEs and TriKEs directly trigger NK cell activation through CD16, significantly increasing NK cell cytolytic activity and cytokine production against tumor targets, demonstrating their therapeutic potential for enhancing NK cell immunotherapies for leukemias and lymphomas. expression of NK cell activating receptor ligands on target cells. In the absence of cytokine stimulation, these receptors inefficiently elicit a cytotoxic or cytokine response independently, but together they are able to function synergistically to activate a resting NK cell and promote effector function (9, 12). In contrast, ADCC is mediated when CD16 binds to opsonized targets through Fc engagement and signals through Rabbit Polyclonal to PTGER3 immunoreceptor tyrosine-based activation motifs (ITAMs) of the associated FcRI chain and CD3 chain subunits (13). The signal delivered via CD16 is potent and induces both a cytotoxic and cytokine response in the absence of cytokine stimulation that can further be enhanced by co-engagement of other activating receptors (12). In this study, we demonstrate the ability of a CD16/CD19 BiKE and a CD16/CD19/CD22 TriKE to trigger NK cell activation through direct signaling of CD16 and induce directed secretion of lytic granules and target cell death. Furthermore, we show for the first time the ability of these reagents to induce NK cell activation that leads to cytokine and chemokine production. Materials and Methods Cell isolation and purification Peripheral blood mononuclear cells (PBMC) were isolated from adult blood (Memorial Blood Center, Minneapolis, MN) by centrifugation using a Histopaque gradient (Sigma-Aldrich) and NK cells were purified by removing T-cells, B-cells, stem cells, 9-Aminoacridine dendritic cells, monocytes, granulocytes and erythroid cells via magnetic beads per the manufacturer’s protocol (Miltenyi Biotec). Primary acute myelogenous leukemia (AML), acute lymphoblastic leukemia (ALL) and chronic lymphoblasitc leukemia (CLL) samples were obtained from the Leukemia MDS Tissue Bank (LMTB) at the University of Minnesota. All samples were obtained after informed consent and in accordance with the Declaration of Helsinki, using guidelines approved by the Committee on the Use of Human Subjects in Research at the University of Minnesota. Flow Cytometry Cell suspensions were stained with the following monoclonal antibodies (mAbs): PE/Cy7-conjugated CD56 (HCD56; BioLegend), ECD-conjugated CD3 (UCHT1; Beckman Coulter), FITC-conjugated CD16 (3G8; BD Biosciences), PE-conjugated CD19 (SJ25C1; BD Biosciences), APC-conjugated CD20 (2H7; BioLegend), FITC-conjugated CD22 (H1B22; BioLegend), PerCP/Cy5.5-conjugated anti-human CD107a (LAMP-1) (H4A3; BioLegend), Pacific Blue-conjugated anti-human IFN- (4S.B3; BioLegend) and AF488-conjugated cleaved caspase-3 (9669; Cell Signaling Technology). Phenotypic acquisition of cells was performed on the LSRII (BD Biosciences) and analyzed with FlowJo software (Tree Star Inc.). Construction, expression and purification of bscFv CD16/CD19 and tscFv CD16/CD19/CD22 reagents Synthesis of hybrid genes encoding the bscFv and tscFv reagents were accomplished using DNA shuffling and DNA ligation techniques as previously described (14). The fully assembled gene (from 5 end to 3 end) consisted of an NcoI restriction site, an ATG initiation codon, the VH and VL regions of anti-human CD16 (NM3E2) (6), a 20-amino acid segment of human muscle aldolase, the VH and VL regions of humanized anti-CD22 (14), humanized anti-CD19 (14) and a XhoI 9-Aminoacridine restriction site. The resultant gene fragment was spliced into the pET21d expression-vector and DNA sequencing analysis 9-Aminoacridine (Biomedical Genomics Center, University of Minnesota) was used to verify that the gene was correct in sequence and cloned in frame. Genes encoding the BiKEs and monospecific scFV controls were created in the same manner. For bacterial protein expression and purification by ion exchange and size exclusion chromatography, methods were used as previously described (14). Proliferation Assay The Burkitts lymphoma Raji cell line (ATCC) was cultured at 37C with 5% CO2 in RPMI-1640 medium (Gibco) supplemented with 10% fetal bovine serum (Gibco). 2104 Raji cells were treated with varying concentrations of CD16/CD19/CD22 or non-specific control CD16/EpCAM and 1 nM.