Patients with cGVHD have a reduced ratio of IL-10⁺ B-cell to IFN-γ⁺ CD4⁺ T cells. PBMCs from patients with cGVHD or no-cGVHD and healthy controls (HC) were stimulated with PMA (50 ng/mL) and ionomycin (250 ng/mL) (Sigma-Aldrich), and Brefeldin A (5 µg/mL) (Sigma-Aldrich) was added for the last 6 hours of the culture. Cells were then stained for the surface expression of CD4 and PI and intracellularly for IFN-γ. (A) Representative flow cytometry plots of IFN-γ staining of gated CD4⁺ T cells from cGVHD and no-cGVHD patients. Frequencies of IFN-γ⁺ CD4⁺ T cells were assessed relative to unstimulated controls. (B) Patients with cGVHD had comparable frequencies (left) and absolute numbers (right) of IFN-γ⁺ CD4⁺ T cells compared with no-cGVHD patients and healthy controls. (C) Dot plots comparing the ratios of IFN-γ⁺CD4⁺ T cells/IL10⁺ CD19⁺ B cells between patients with or without cGVHD. Values are medians, with interquartile ranges indicated by brackets (n = 11 per group); *P < .05 by 2-tailed nonparametric Student t test analysis.