2010B080701074). Availability of data and materials The datasets used and/or analyzed during the present study are available from the corresponding author on reasonable request. Authors’ contributions YL was responsible for the experiment and wrote the manuscript. FcRIIIa: FcRIIIa-158 F/F, FcRIIIa-158 V/V and FcRIIIa-158 V/F. Polymorphisms (VV/VF/FF) of FcRIIIa markedly affect the binding affinity of mAbs, particularly the IgG1 isotype, and has been observed in multiple malignancies by its association with the degree of lysis of tumor cell targets (15C17). Normal serum levels of IgG may effectively compete with IgG1 for binding to low-affinity FcRIII (CD16) (28), we collected 5 ml of peripheral blood from healthy volunteers, and coagulation was allowed for 20 min followed by centrifugation of the collection tubes. Immediately after centrifugation, serum was aliquoted in 1.5-ml polypropylene tubes and frozen at ?20C until use. When processing, serum was defrosted and diluted in 1:1 with RPMI 1640, producing a medium with 50% human serum (containing complement). Serum IgG Serum was obtained by centrifugation of peripheral blood. Complement in the serum was inactivated in a 56C water bath incubator for 30 min (28). The inactivated serum was mixed with RPMI 1640 at a ratio of 2:3, achieving a medium of 40% human serum (containing serum IgG). Serum IgG and FcRIIIa binding assays in the absence of mAb The binding of serum IgG to FcRIIIa on NK cells was studied TFMB-(R)-2-HG by flow cytometry. Briefly, 0.1 ml of 5106/ml PBMNCs were incubated in the absence or presence of 4.8 mg/ml human serum IgG for 30 mins at 37C in a 5% CO2 incubator, washed twice with PBS, followed by flow cytometric analysis of cell-bound FcRIIIa. Cytotoxicity assay The cytotoxicity assay was divided into two groups (FcRIIIa V/V and FcRIIIa V/F) according to the FcRIIIa genotypes of NK cells, and each group was further subdivided into four groups: Negative control, ADCC, ADCC+CDC and serum IgG groups. Raji cells were labeled with DIO (Beyotime Biotechnology, Jiangsu, China) at 37C for 30 min and washed three times with PBS to remove unreacted and unbound DIO. A total of 3 l of 0.1 g/l rituximab was added to the ADCC, ADCC+CDC and serum IgG groups, and serum was added to the serum IgG group at the same time. Each group was incubated for 4 h at 37C in a 5% CO2 incubator, and then the labeled target cells were re-suspended in RPMI 1640 containing 10% FCS (only the ADCC+CDC group was re-suspended in RPMI 1640 containing 50% human serum) and mixed with PBMNCs at an effector/target ratio (E/T) of 5:1. All the cells were incubated at 37C for 4 h and washed twice with PBS, followed by the addition of 5 l propidium iodide (PI) (Beyotime Biotechnology). Finally, cells were analyzed by flow cytometry after a 30-min incubation in the dark. The negative control did not contain PBMNCs. The percentage of killed cells was calculated as follows: (% of living cells in negative control-% of living cells in sample)/% of living cells in negative control. Statistical analysis The results are expressed as the mean standard error of the mean, and the data were TFMB-(R)-2-HG analyzed by SPSS 16.0 statistical software. An independent samples t-test was used to evaluate the difference between FcRIII-positive PBMNC and FcRIII-positive NK cells in PBMNCs. The expression levels of FcRIIIa in NK cells before and after adding serum in the absence of mAb were also analyzed using an independent samples t-test. The comparison of cytotoxic ITGAM index between the groups with multivariate analysis of variance, after the equal check TFMB-(R)-2-HG of variance, and the two-two comparisons among the means were performed using the Student-Newman-Keuls method. P 0.05 was considered to indicate a statistically significant difference. Results Human PBMNCs may be an alternative to NK cells as the effector cells In this study, the results demonstrated that 20.912.12% of PBMNCs were CD3?D56+ NK cells (Fig. 1A), and the expression level of FcRIII on NK cells was 91.296.53% (Fig. 1B). A total of 19.240.78% of PBMNCs expressed FcRIII (Fig. 1C), and NK cells expressing FcRIII accounted for 19.020.57% of PBMNCs (Table I and Fig. 2); thus, NK cells were the main FcRIII-positive cells in PBMNCs. Therefore, FcRIII-positive PBMNCs may be an alternative to FcRIII-positive NK cells as effector cells in our experiment, which was also confirmed by other studies (25C27). Open in a separate window Figure 1. Surface markers of NK cells, PBMNCs, CD3,.