Presence of DBY/HLA class II complex antibody is a risk factor for cGVHD development in F-to-M HCT. (A) Flow cytometry of plasma antibodies binding to DBY complexed with HLA class II molecules in patients after F-to-M HCT. Diluted plasma was incubated with 293T cells transfected with mock (shaded histogram), HLA-DRB1∗15:02 (blue line), or DBY and HLA-DRB1∗15:02 (red line). IgG antibody binding to the transfected cells was assessed by staining with antihuman IgG antibody. Patients 1 to 6 (upper 6 histograms) did not develop cGVHD, whereas patients 7 to 12 (lower 6 histograms) developed cGVHD after F-to-M HCT. (B) Comparison of the titers of anti-DBY/HLA class II complex antibodies between F-to-M and other HCTs (M-to-M, M-to-F, and F-to-F) at 3 months after allo-HCT. The cutoff value was determined by the 95th percentiles of MFIs, except for F-to-M HCT. The dashed line shows the cutoff value for the MFIs. Individuals who underwent M-to-M, M-to-F, or F-to-F HCT are indicated by blue circles, whereas those who underwent F-to-M HCT are indicated by orange circles. (C) Cumulative incidence of cGVHD based on the presence of anti-DBY/HLA class II complex antibodies after F-to-M HCT. The Gray test was used to compare the anti-DBY/HLA class II complex antibody–positive (red line) or –negative (blue line) groups. (D) Relationships between the titers of anti-DBY/HLA class II complex antibodies and those of anti-DBY antibodies. The cutoff value was determined by the 95th percentiles of the MFIs and optical density (OD) values, except for F-to-M HCT. Dashed lines show the cutoff values for the MFIs and OD values. The circles indicate individuals. Mock: pSI vector.