Fig. 5

Serum IL-39 levels were significantly elevated in patients with cGVHD. CXCL13 and IL-39 levels in serum were detected in non-GVHD, mild, and moderate/severe cGVHD patients (A). The prognostic value of CXCL13 and IL-39 in no-cGVHD and mild and moderate/severe cGVHD patients was evaluated using ROC curves (B). CD4⁺T cells, CD8⁺T cells, B cells, and CD11b⁺ cells were sorted from the splenocytes of C57BL/6 mice or PBMCs from healthy donors using a Cell Isolation Kit according to the manufacturer’s protocol. For CD4⁺ and CD8⁺T cell activation, plates were coated with 2 mg/ml anti-CD3 and 0.4 mg/ml anti-CD28 Abs overnight. Next, 2 × 10⁵ T cells were cultured with recombinant mouse or human IL-39 proteins (20 ng/ml) for 72 h. For B cell and CD11b⁺ cell activation, 2 × 10⁵ B cells and CD11b⁺ cells were cultured with LPS (5 ng/ml) and recombinant mouse or human IL-39 proteins (20 ng/ml) for 72 h. The relative expression of IL-39 and IL-39R genes was determined by real-time PCR in CD4⁺T cells, CD8⁺T cells, B cells, and CD11b⁺ cells from the splenocytes of C57BL/6 mice and PBMCs from healthy donors (C). In the following experiments, plates were coated with 2 mg/ml anti-CD3 and 0.4 mg/ml anti-CD28 Abs overnight. T cells (2 × 10⁵ T cells were cultured with various concentrations of recombinant mouse or human IL-39 proteins for 72 h. Phosphorylation of STAT1/STAT3 detected by western blotting in sorted primary T cells from mice or healthy donors is shown (D, E). The data are representative of at least three independent experiments. Values are presented as mean ± SEM. *P < 0.05; ***P < 0.001