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. 2020 Dec 22;11:606518. doi: 10.3389/fimmu.2020.606518

Figure 6.

Figure 6

αRP105-TM can activate both mature and immature B cells. (A) HEK293T cells were transfected with pCADEST1-empty (Vector), pCADEST1-anti-OVA kappa-F2A-anti-OVA mIgG1/TM [Isotype-2-TM (F2A)], or pCADEST1-anti-RP105 kappa-F2A-anti-RP105 mIgG1-TM [αRP105-TM (F2A)]. Two days later, the splenocytes obtained from BALB/c mice were incubated with the HEK293T cells for 7 days. The supernatant was obtained, and the whole IgG level was measured by quantitative ELISA. The detection limit was over 3.91 ng/ml. ***P < 0.001 (One-way ANOVA). (B, C) HEK293T cells were transfected as indicated. Two days later, splenocytes obtained from BALB/c mice were co-cultured with HEK293T cells for 2 days. Then, the splenocytes, which were gated on B220 (B), IgMlow IgDhigh as mature B cells (Middle panel in C), or IgMhigh IgDlow as immature B cells (Right panel in C), were also incubated with biotinylated parental anti-RP105 mAb (rat) or anti-CD86 antibodies as indicated, followed by incubation with PE-conjugated streptavidin. The indicated numbers on each gate (Left panel in C) respectively represent the percentage of cells of mature or immature B cells gated on B220+ cells. The numbers in the histogram represent MFI. All indicated data are representative of two independent experiments.