Improved total IgG and antigen-specific IgG responses in RG SKI IL-6 mice. (A) Concentration of total hIgM and hIgG in sera. (B) Humanized mice were immunized with OVA as described in “Materials and methods.” Concentration of total hIgM and hIgG in sera were shown after immunization. The data represent the mean ± SEM (n = 17-22 per group). (C) Spleen cells were collected and analyzed after immunization. Gates used for identification of human memory, IgG⁺, immature, transitional, or plasmablast B cells by flow cytometry based on the expression of hCD27, hIgG, hCD10, hCD24, and hCD38 (top). The relative abundance of each B-cell subset is compared between two mouse strains (bottom). (D) Gates used for identification of CD27⁻IgG⁺ and CD27⁺IgG⁺ B cells (top). The relative abundance of each cell subset is compared between two mouse strains (bottom). (E) Representative ELISA results from humanized mice with serum sample dilutions of 1:100. Data were presented as normalized absorbance ratio by dividing the OD value of antigen-specific IgG with the OD value of total IgG in each mouse to exclude the variation between the individual animals. The dotted line represents the cutoff value (cut off value = mean OD values of the negative control + 2 × SD). Negative control is the mean OD value of OVA-specific IgG of RG SKI and RG SKI IL-6 mice before the immunization. (F) Histologic examination of spleen tissues from engrafted mice. Sections were stained with hematoxylin and hCD20 (stained in red). All the data were analyzed by unpaired Student t test. *P < .05; **P < .01; ***P < .001. OD, optical density; SD, standard deviation.