Figure 1.

Antigen binding of germline reverted TII AVAs. Nine somatically hypermutated (mut) AVAs and their corresponding predicted germline reversions (rev) were assayed for autoreactivity. (A) Reactivity with recombinant vimentin or BSA were measured by ELISA (Raw OD₄₀₅ values are given and t-tests were performed between mut and rev variants of respective AVAs). (B) Representative HEp-2 multi-color immunofluorescence microscopy images stained with indicated mutated and reverted TII antibodies with vimentin (V9) and nucleus (Hoechst). Representative of 3 independent experiments. (C) HEp-2 multi-color immunofluorescence microscopy method for quantification of relative AVA reactivity with different subcellular areas. Individual raw channels including DIC were used for segmenting whole cells and subcellular areas. Areas high in reactivity with Hoechst and V9 were autothresholded and designated nuclear (“nuc”) and vimentin^high (Vim^hi) respectively. Cell perimeters were manually segmented. Representative example provided. (D) Relative reactivities (measured as mean pixel intensities, MPI) of AVAs with different subcellular areas. Each dot represents the MPI in the AVA channel for the specified subcellular zone of an individual cell. Pairwise comparisons (Wilcoxon matched-pairs tests) were performed between MPIs of Vim^hi and indicated subcellular areas. (E) Co-variances of pixel intensities were quantified between the AVA channel and either V9 or Hoechst channels. Dots represent correlation coefficients between pixel intensities for multicellular fields of view (FOV). Mann-Whitney tests were performed between somatically hypermutated (mut) and germline reversion values. For 1d, binding in each group was compared to vimentin binding. na, not applicable; ns, not statistically significant. For panel A, q values were calculated, *q < 0.05, **q < 0.01, ***q < 0.001. Other values are p values, same key. Red scale bar = 10 microns.