Background

Allo-HSCT is an effective method for the treatment of hematologic malignancies. However, cGVHD is a major complication of allo-HSCT, occurring in approximately 50% of patients [1,2,3,4]. Similar to aGVHD, donor T cells play a critical pathogenic role in initiating tissue injury and develo** cGVHD [5]. Depleting mature T cells from stem cell grafts markedly reduces the incidence of cGVHD [6,7,8,9]. In addition to T cell activation, autoreactive B cells have also been found to play a pathogenic role in cGVHD. Under the action of high-level B cell activation factor (BAFF), more donor B cells differentiate into autoantibody-secreting subsets upon sustained stimulation of receptor antigens, resulting in pathological organ damage [10,11,12]. Therefore, exploring key molecules that cause immune dysregulation in T and B cells is expected to provide new mechanisms and targets for cGVHD prevention and treatment.

The IL-12 family consists of IL-12 (IL-12p35/IL-12p40), IL-23(IL-23p19/IL-12p40), IL-27 (IL-27p28/EBi3), IL-Y (IL-27p28/ IL-12p40), and IL-35 (IL-12p35/EBi3) [13, 14]. IL-12 and IL-23 play crucial roles in inducing differentiation of Th1 and Th17 cells, respectively [15]. IL-Y promotes cGVHD by activating pathogenic T and B cells. In contrast, IL-27 and IL-35 suppress inflammatory responses by promoting the expansion of regulatory B and T cell subsets [13, 15,16,17]. Wang et al. first described a new IL-12 member composed of IL-23p19 and an EBi3 heterodimer, IL-39 [Mice

6–8-week-old female C57BL/6 (H-2b) and BALB/c (H-2d) mice were purchased from SLAC Animal Laboratory (Shanghai, China). 8–10-week-old female DBA/2 mice (H-2d) were purchased from Charles River Laboratories (Bei**g, China). The experimental animals were kept under specific pathogen-free (SPF) conditions.

Establishment of murine models of cGVHD

One week before setting up the experiments, recipient mice were fed gentamicin aqueous solution to prevent intestinal infections. For HGT, recipient mice (BALB/c) were injected i.v. with 120 μg of recombinant plasmid in a total of 2 ml PBS within 5 s at 3 days before transplantation. Recipient BALB/c mice were given total body irradiation (TBI) at 650 cGy using a RAD 320 X-ray Irradiator 6–8 h prior to transplantation. To establish a scleroderma-like cGVHD model, recipients were infused with 1 × 107 bone marrow cells and 1 × 106 splenocytes from the C57BL/6 mice. To establish a lupus-like cGVHD model, irradiated recipients (BALB/c mice) were infused with 5 × 106 bone marrow cells and 4 × 107 CD25− splenocytes from DBA/2 mice. To investigate the preventable effect of anti-IL-39 on cGVHD, 14 days after transplantation, mice received 100 μl (100 μg) of anti-IL-39 antibody (DETAIBIO, China) or 100 μl (100 μg) of non-specific IgG (BioXcell, BE0095) via intraperitoneal injection twice a week for 6 weeks.

Histology

Representative samples of the lung, liver, small intestine, skin, and kidney were obtained from transplanted recipients, fixed in 4% formalin, and stained with H&E. The pathology score of the target organs was based on a previous scoring system [40, 41].

Flow cytometry

The antibodies used for flow cytometry are listed in Additional file 1: Table S3. For intracellular staining, cells were treated with brefeldin A (BFA, 10 μg/ml) (Biolegend, San Diego, CA), phorbol-12-myristate-13-acetate (PMA, 50 ng/ml) (Beyotime Biotechnology, China), and ionomycin (500 ng/ml) (Beyotime Biotechnology, China) in a 37 °C cell culture incubator containing 5% CO2. The cells were then fixed and permeabilized with FACS Permeabilizing Solution (BD Biosciences, San Diego, CA, USA). Foxp3 staining kit was purchased from eBioscience (San Diego, CA, USA). Data were acquired using FACS NovoCyte (ACEA Biosciences, San Diego, CA, USA) and analyzed using FlowJo software (FlowJo, Ashland, OR, USA).

Patients and sample preparation

Fifty patients who underwent hematopoietic stem cell transplantation at the First Affiliated Hospital of Soochow University between April 2015 and August 2017 were enrolled. We grouped the patients into no cGVHD, mild cGVHD, and moderate/severe (M/S) cGVHD groups according to the NIH consensus criteria [42]. The characteristics of the patients are shown in Additional file 1: Table S2. Peripheral blood samples obtained from 8 patients without cGVHD after HSCT were used as controls. The levels of IL-39 (RapidBio, USA) and CXCL13 (R&D Systems, UK) in serum samples were detected using an enzyme-linked immunosorbent assay (ELISA).

Real-time quantitative polymerase chain reaction (qPCR)

Murine CD4+T cells, CD8+T cells, and B cells were sorted from splenocytes of C57BL/6 mice, whereas human CD4+T cells, CD8+T cells, and B cells were sorted from PBMCs of healthy individuals using CD4+T, CD8+T, and B Cell Isolation Kits according to the manufacturer’s protocol (StemCell Technologies, Vancouver, Canada). Similarly, CD11b+ cells were sorted from splenocytes of C57BL/6 mice or human PBMCs using CD11b micromagnetic beads, according to the manufacturer’s protocol (Miltenyi Biotec, Germany). For T cell activation, plates were coated with 2 mg/ml anti-CD3 and 0.4 mg/ml anti-CD28 Abs (BioLegend, San Diego, CA) overnight. 2 × 105 CD4+T and CD8+T cells (2 × 105) were cultured with various concentrations of mouse rIL-39 for 72 h at 37 °C in a cell culture incubator containing 5% CO2. For B cell and CD11b+ activation, 2 × 105 B cells and CD11b+ cells were cultured with LPS (5 ng/ml) and various concentrations of rIL-39 protein for 72 h. Consistently, activated CD4+T, CD8+T, B, and CD11b+ cells isolated from the PBMCs of healthy individuals were cultured with various concentrations of human rIL-39 for 72 h. Total RNA was isolated using TRIzol Reagent (Invitrogen, Carlsbad, CA, USA), according to the manufacturer’s instructions. cDNA was synthesized using reverse transcription, random hexamer primers, and 10 mM dNTP (Promega). The expression of EBi3 and IL-23p19 in the spleen at 10 days and in the liver, lung, and intestine at 30 days post-HSCT was regarded as control. The relative expression of genes in one organ at the remaining time point was calculated by 2−ΔΔCt according to the control. The primer sequences are listed in Table S1.

Western blotting analysis

Murine CD3+T cells were sorted from splenocytes of C57BL/6 mice, and human CD3+T cells were sorted from peripheral blood mononuclear cells (PBMCs) of healthy individuals using a T Cell Isolation Kit, according to the manufacturer’s protocol (StemCell Technologies, Vancouver, Canada). For T cell activation, plates were coated with 2 mg/ml anti-CD3 and 0.4 mg/ml anti-CD28 Abs (BioLegend, San Diego, CA) overnight. Activated T cells were cultured with various concentrations of mouse or human rIL-39 protein for 72 h. Equal amounts of protein were subjected to 10% SDS-PAGE and transferred to a polyvinylidene fluoride (PVDF) membrane. After blocking with 3% BSA, the membrane was incubated with primary antibodies, including STAT1 (D1K9Y), Phospho-STAT1 (58D6), STAT3 (D3Z2G), Phospho-STAT3 (D3A7), and GAPDH (D4C6R) (CST, USA) at 4 °C overnight, followed by incubation with secondary antibodies (Absin, China) at room temperature for 2 h. All results were normalized to GAPDH expression, which was used as the loading control.

Statistical analysis

Data were presented using GraphPad Prism 7 software (GraphPad Software, San Diego, CA, USA). The nonparametric Mann–Whitney U test was used to analyze the body weight and clinical scores between the groups. Comparisons between two groups were performed using unpaired two-way Student’s t-tests. The diagnostic value of biomarkers was evaluated using ROC curves. Data are expressed as the mean ± SD or SEM. For all statistics, if P < 0.05, they were considered statistically significant (*), less than 0.01 or 0.001 were shown as ** or ***, respectively.