Affinity maturation generates pathogenic antibodies with dual reactivity to DNase1L3 and dsDNA in systemic lupus erythematosus


Fig. 6. Characterization of pathogenic and non-pathogenic SLE-derived monoclonal antibodies categorized as anti-dsDNA.

a Ig gene usage, mutation number and CDR3 amino acid sequences of monoclonal antibodies 32.B9, 33.H11, 33.C9, and RH-14. bd Mutated and germline reverted (GL) antibodies 32.B9, 33.H11, 33.C9, and RH-14 were titrated against DNase1L3 (b), dsDNA (c) and cardiolipin (d). Curves on the graph correspond to the fitted four-parameter logistic (4PL) model used to calculate the EC50. e Comparison of the EC50 for antibody binding to DNase1L3 (yellow), dsDNA (green), and cardiolipin (pink) using mutated (32.B9, 33.H11, 33.C9, and RH-14) and GL reverted (32.B9GL, 33.C9GL and 33.H11/RH14GL) monoclonal antibodies. Monoclonals 33.H11 and RH-14 are the same antibody when reverted to GL. Bars show the mean EC50 and the error bars represent upper and lower EC50 values. f Effect of monoclonal antibodies (C4 control, 32.B9, 33.H11, 33.C9, and RH-14) on DNase1L3 activity. Chromatin degradation was quantified by LM-qPCR as described in Fig. 4d. Experiments were performed in two separate occasions (bd, f). g Radiolabeled DNase1L3 was immunoprecipitated (IP) in the absence (−) or presence (+) of salmon sperm DNA (1 mg/mL) using representative SLE sera positive for antibodies to DNase1L3. Relative quantification of IP inhibition was calculated by the ratio of the densitometric value of sera with/without DNA.

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