Figure S1.

Isolation and generation of pathogenic monoclonal AIGAs from patients underlying mycobacterial diseases. (A) Identification of AIGAs in patient plasma (n = 3) by ELISA. Bar graph of antibody binding to IFN-γ (2 μg/ml) in several dilutions of plasma (100×, 500×, and 2,500×). Each dot corresponds to a donor. (B) HLA-DRB1 and -DQB1 alleles in recruited patients with AIGAs. (C and D) Evaluation of the biological function of AIGAs from three recruited patients. (C) Amount of IFN-γ detected in whole-blood activation assays (NA, BCG, and BCG + 20 ng/ml IL-12) on blood samples from the corresponding donors. Each dot corresponds to a donor. (D) Amount of IL-12p40 detected in whole-blood activation assays (NA, BCG, and BCG + 250 ng/ml IFN-γ) on blood from the corresponding donors. Each dot corresponds to a donor. (E) Isolation of single IFN-γ–specific IgG⁺CD19⁺ B cells from patients with mycobacterial disease. Representative flow cytometry plots showing the gating strategy for human IFN-γ–specific B cells from the PBMCs from three patients. Black boxes indicate each successive gate used. Lymphocytes, identified on the basis of forward and side scatter (FSC and SSC), were further analyzed by FSC-A vs. FSC-H comparisons and with 7-aminoactinomycin D staining to eliminate doublets and dead cells. CD19⁺CD3⁻ cells were then selected, followed by IgG⁺IgD⁻ cells, to obtain enrichment in specific B cells binding to IFN-γ, which were stained with a FITC-conjugated antibody. IFN-γ⁺ cells were then sorted from IgG⁺ subsets. (F) Bar graph showing the reactivity of the 19 monoclonal AIGAs and AMG811 (1 μg/ml) with recombinant human IFN-γ (2 μg/ml), as determined by ELISA. The results are shown as the mean and SD for two independent experiments. (G) Western blot showing the reactivity of 19 monoclonal AIGAs, AMG811, and control IgG (ctrl.; 1 μg/ml) with the recombinant human IFN-γ protein (300 ng/well). The experiments were performed twice, independently. Source data are available for this figure: SourceData FS1.