Natural IgG3 is secreted independently of Blimp-1. (A) Shown are serum Ig isotype concentrations ± SD (mg/ml) in control and PRDM-1^ΔEx1A mice (n = 5–9 for IgA, IgG1, IgG2c, and IgG2b; n = 12–15 for IgG3). Results are pooled from two independent experiments. (B) Mean frequencies ± SD of IgG3 ASCs among the spleens and BM from control and PRDM-1^ΔEx1A mice (n = 3). (C, left) Mean spot sizes ± SD (number of pixels) from IgG3 ASCs in the spleens of control and PRDM-1^ΔEx1A mice (n = 3) and (C, right) representative ELISPOT wells. Numbers indicate total input cells. Results in B and C are representative of two independent experiments. (D) Serum IgG3 (mg/ml) ± SD in mice with only B-1 cells (neonatal chimeras, day 42, during DS-1 treatment) and in the same mice after reconstitution of B-2 cells (day 94). Each line represents one mouse. Results are combined from two independent experiments. (E, left) Sample gating for total IgG3⁺ and IgG3^neg cells, as well as IgG3⁺ Blimp-1 YFP⁺ and YFP^neg cells, pregated on live, singlet, and dump^neg cells. FMO, fluorescence minus one control stains for IgG3. Values shown in FACS plot represent percentage within parent population. Cells were sorted directly into ELISPOT plates. (E, right) Mean frequencies ± SD of IgG3 ASCs among splenic IgG3⁺ (total), IgG3⁺ Blimp-1 YFP⁺ (YFP+), and IgG3⁺ YFP^neg (YFP-) and IgG3^neg cells (n = 4–8). The dashed line represents the limit of detection. (F, left) Mean spot sizes ± SD (number of pixels) from splenic IgG3⁺ Blimp-1 YFP⁺ and IgG3⁺ YFP^neg cells (n = 3) and (F, right) representative ELISPOT wells. Results in E and F are representative of three independent experiments. Statistics in A–C and F were done using an unpaired Student’s t test (*, P < 0.05).