Loss of B-cell tolerance in IPEX mice. (A) Serum immunoglobulin levels quantified by ELISA from 2 independent experiments. (B) Autoantibody detection by indirect immunofluorescence on cryosections of frozen lung and liver tissue from a nonhumanized NSGAb°DR1 mouse. Frozen sections were incubated independently with sera from 4 individual reconstituted mice per group. Fluorescein isothiocyanate (FITC)-conjugated anti-human IgG was used as a secondary antibody combined with DAPI to label nuclei. (a) Denotes lung airway. Scale bars are 50 μm. (C) Heat map of antibody response to select murine antigens determined using an antigen microarray. Each column is the IgM and IgG serum reactivity from an individual mouse in each group with the averaged reactivity score for all antigens per group noted at the bottom. IgM P = .06, IgG P = .09 (D) The normalized cumulative autoreactive IgM and IgG antibody reactivity values for all 500 mouse antigens in arbitrary units (a.u.) were plotted against the % human B cells in each mouse (n = 5). Solid line marks the best-fit linear regression for each cohort with the R2 value calculated using Pearson correlation coefficients. For each data point, the % of human chimerism is shown in parenthesis and for IPEX(DR1) mice, the age in weeks when mice became moribund and were killed. Statistical analysis was performed using Mann-Whitney test (A) and nonlinear regression correlation using Pearson correlation coefficients (D). ***P < .001.
