Figure 7.

Developmental sites and stages of B cell immune tolerance. (A) Comparison of dsDNA and HEp-2 cell binding of antibodies representing different stages of selection in the Ig^Vκ4/h mice. All antibody binding data for HEp-2 cell and dsDNA by ELISA and the blinded scores for anti–HEp-2 IMF were compiled to compare the relative levels of autoreactivity before and after B cell selection. To do so, preselection recombinant mAbs composed of VH genes from allelically included (Vκ4κ⁺hCκ⁺) or allelically excluded (Vκ4κ⁻hCκ⁺) edited mature cells paired with the Vκ4 LC and postselection recombinant mAbs composed of VH genes from mature unedited cells (Vκ4⁺hCκ⁻) paired with the Vκ4 LC or VH genes from mature edited cells (Vκ4⁻hCκ⁺) with the hCκ edited LC were compared. Recombinant antibodies were made with VH genes from 12 Vκ4⁻hCκ⁺ mature cells, 32 Vκ4⁺hCκ⁺ mature cells, 62 postselection mature Vκ4⁺hCκ⁻ cells, and 44 Vκ4κ⁻hCκ⁺ edited mature cells. (B–E) Autoreactivity of mAbs cloned from single-cell sorted mature (AA4⁻) unedited Vκ4⁺hCκ⁻ B cells from the spleens of Igκ^αhel/h and Igκ^Vκ4/h mice. Antibodies were generated from a total of 37 B cells from three Igκ^Vκ4/h mice (n = 10, 10, and 17 mAbs by mouse) and a total of 34 B cells from three Igκ^αhel/h mice (n = 11, 7, and 16 mAbs by mouse). The frequency of autoreactivity was tested and analyzed as in Figs. 2 and 3 using HEp-2 cells and anti-dsDNA ELISAs (B–D) and by HEp-2 IMF (E). In E, the results of blinded scoring on a scale of 0–4 are shown in pie charts. All assays were repeated at least three times. Statistic used was the χ² test p-value for E. (A, C, and D) Red lines indicated the mean AUC.