To avoid killer cell immunoglobulin-like receptor (KIR) dependent ADCC, HLA class I expressing (ARH-77) and nonexpressing (Daudi) cell lines (both of which are CD20+) were used like a target cells. better medical outcomes due to increased CD16 manifestation, rituximab binding, and rituximab-mediated ADCC. Intro Rituximab is definitely a CD20-directed, IgG1-chimeric monoclonal antibody (mAb) used to treat individuals with B-cell lymphomas and various autoimmune disorders. Both quantitative as well as qualitative variations in natural killer Syncytial Virus Inhibitor-1 (NK) cell function may clarify rituximab medical activity. Higher circulating NK cell levels and reactions to rituximab have been reported in individuals with indolent non-Hodgkin lymphoma (NHL), suggesting that antibody-dependent cellular cytotoxicity (ADCC) enacted by NK cells may be a primary mechanism by which rituximab functions.1,2 Moreover, reactions to rituximab may depend upon polymorphisms present in the FcRIIIa (CD16) receptor, a receptor mainly expressed on NK cells.3C5 Polymorphisms in position 48 and 158 of the FcRIIIa receptor expression have been reported to influence human IgG1 binding and ADCC activity.6C9 Polymorphisms at position 158 result in either valine (V) or phenylalanine (F) expression,6,8,9 the former of which is associated with increased depletion of peripheral blood B cells10 and response to rituximab in patients with indolent NHL3C5 but not chronic lymphocytic leukemia (CLL).11 At position 48, polymorphisms of the FcRIIIa receptor result in expression of either leucine, arginine, or histidine, the first of which is linked to FcRIIIa-158F and the second option 2 with the FcRIIIa-158V polymorphisms.5,8,9 However, the binding of IgG1 to FcRIIIa appears to happen independently of position 48 polymorphisms most likely on the basis of limited genetic linkage to FcRIIIa-158 polymorphisms.5,8 Genetic linkage between polymorphisms in FcRIIa (CD32), a receptor also implicated in predicting rituximab clinical response, and FcRIIIa has recently been demonstrated by us and points to the primacy of FcRIIIa-158 polymorphisms in predicting rituximab response.12 While these studies suggest that variable reactions to rituximab among FcRIIIa-158 polymorphic organizations are likely the result of qualitative (ie, antibody affinity) differences, the possibility that quantitative differences in cell surface CD16 manifestation, rituximab binding, and ADCC activity have not been addressed. As such, we wanted to delineate variations in FcRIIIa gene manifestation, cell surface CD16 manifestation, rituximab binding, and rituximab-dependent ADCC activity in NK cells isolated from healthy individuals representing the 3 FcRIIIa-158 polymorphic subgroups (V/V, V/F, and F/F). Materials and methods FcRIIIa-158 genotype analysis We analyzed the genotype of 52 unrelated healthy individuals by sequencing exon 4 of the FcRIIIa gene. FcRIIIa-158 polymorphisms were determined by allele-specific reverse transcription polymerase chain reaction (RT-PCR) and direct sequencing of genomic DNA, as we previously described.5 Genomic Syncytial Virus Inhibitor-1 DNA was extracted from peripheral blood using a DNA isolation kit (Qiagen, Valencia, CA). The study was authorized by the Dana Farber Malignancy Institute’s Institutional Review Table, and written consent was from each donor in accordance with the Declaration of Helsinki. Cell isolation and tradition Peripheral blood mononuclear cells (PBMNCs) were isolated using Ficoll-Paque (Amersham, Uppsala, Sweden). NK cells were selected from PBMNCs using the NK-cell isolation kit II (Miltenyi, Auburn, CA) resulting in more than 95% purity (CD3?/CD56+). ARH-77 and Daudi cells were cultured as previously explained. RT-PCR analysis FcRIIIa gene manifestation was determined by quantitative real-time RT-PCR (Applied Biosystems, Foster City, CA). RNA was extracted from NK cells. Primer sequences were as follows: FcRllla sense (5-CCAAAAGCCACACTCAAAGAC-3) and antisense (5-ACCCAGGTGGAAAGAATGATG-3); TaqMan probe (5-AACATCACCATCACTCAAGGTTTGG-3). The amount of FcRIIIa mRNA in each sample was normalized to the relative quantity of HR-18S. Quantitative circulation cytometry CD16 receptors were quantified using the QuantiBRITE system. NK cells Syncytial Virus Inhibitor-1 (2 105) were stained with 5 L (0.287 mg/mL) of anti-CD16 PE bead-conjugated mAb for 20 minutes at 4C (BD Biosciences, San Jose, CA). After incubation, NK cells were washed twice and resuspended in 1 PBS. Prior to each analysis, the circulation cytometer was calibrated by QuantiBRITE PE calibration beads. CD16 receptors were assessed by gating 104 (CD3?CD56+) cells. Samples were FLJ16239 analyzed using CellQuest software (BD Biosciences). Rituximab binding to NK cells Rituximab (Genentech BioOncology, San Francisco, CA) binding was identified using an indirect method as previously explained,7 using an anti-CD16 (3G8 clone) mAb. NK cells (2 105) were incubated with rituximab at concentrations of 10, 50, 100, and 200 g/mL for 30 minutes.