Unique antigen targets of IgG in active cGVHD plasma include the autoantigen Ro-52, which requires RNA for antibody binding. (A) Plasma samples from patients (n = 10) with active cGVHD along with pre-HSCT and corresponding donor samples were screened by plasma microarray (ProtoArray) for reactivity against 8000 target human proteins. To ensure consistency between samples, all 30 arrays (n = 10 pre-HSCT, n = 10 cGVHD, and n = 10 donor, all corresponding to the appropriate individual receiving transplantation) were from the same lot and the process was performed by the same operator. Antigens that were uniquely targeted after development of cGVHD were identified by eliminating those that overlapped among cGVHD samples and corresponding pre-HSCT or donor samples. Proteins targeted by >1 cGVHD plasma sample are highlighted in gray. (B) anti–Ro-52 IgG responses noted on protein microarray in plasma from patients with cGVHD as compared with corresponding donor and pre-HSCT plasma samples were confirmed via ELISA. (C-D) Plasma was isolated serially from patients over a period of 12 months post HCST and anti-Ro-52 IgG antibodies were detected by direct binding ELISA in those who (C) did develop (n = 15-18) or (D) did not develop (n = 5-8) cGVHD symptoms. Dotted line represents assay threshold of positivity based on values from healthy controls. (E) B cells from a patient whose plasma was positive for anti–Ro-52 IgG were sorted on the basis of IgD and CD38 expression. Cells were cultured with EBV ± BAFF at 100 cells per well. After 21 days, B-cell supernatants were collected from individual wells and levels of anti–Ro-52 IgG were assessed via ELISA. Each mark represents 1 of 96 wells of IgG⁺ supernatant from B-cell cultures. (F) anti–Ro-52 IgG levels in the 2 patients with cGVHD who were anti–Ro-52 positive identified by ProtoArray before and after RNase treatment as determined by direct-binding ELISA.