Construction of PD-L1hitol-DC derived from bone marrow of DA rats and identification of its immunological function
10.12464/j.issn.1674-7445.2024183
- VernacularTitle:DA大鼠骨髓来源PD-L1hitol-DC构建及其免疫学功能鉴定
- Author:
Zhiqi YANG
1
;
Peibo HOU
1
;
Lang WU
1
;
Jing LIU
1
;
Yang DING
1
;
Minghao LI
1
Author Information
1. Department of Hepatobiliary Surgery, People＇s Hospital of Ningxia Hui Autonomous Region, Ningxia Medical University, Yinchuan 750000, China.
- Publication Type:OriginalArticle
- Keywords:
Dendritic cell;
Organ transplantation;
Immune tolerance;
Immunosuppression;
Programmed cell death protein-ligand 1;
T cell;
Regulatory T cell;
Inflammatory factor
- From:
Organ Transplantation
2025;16(1):83-90
- CountryChina
- Language:Chinese
-
Abstract:
Objective To construct programmed cell death protein-ligand 1(PD-LI)hi tolerogenic dendritic cell (tol-DC) derived from bone marrow of DA rats and identify its immunological function. Methods DA rat bone marrow cells were extracted, combined with recombinant mouse granulocyte macrophage colony-stimulating factor and recombinant mouse interleukin (IL)-4, and cultured for 6 days in vitro to induce the differentiation of bone marrow cells into immature dendritic cells (imDC). Lipopolysaccharide was used to stimulate cell maturation and cultured for 2 days to collect mature dendritic cells (mDC). PD-L1 lentiviral vector virus stock solution or equivalent dose lentiviral stock solution was added, and PD-L1hitol-DC and Lv-imDC were collected after culture for 2 days. The morphology of PD-L1hitol-DC was observed by inverted phase contrast microscope and transmission electron microscope. Real-time fluorescence quantitative reverse transcription polymerase chain reaction, Western blotting and flow cytometry were used to detect the expression level of specific markers on cell surface. CD8+T cells derived from Lewis rat spleen were co-cultured with imDC, mDC, Lv-imDC and PD-L1hitol-DC, respectively. The levels of inflammatory factors in the supernatant of each group were detected by enzyme-linked immunosorbent assay. The apoptosis of T cells and the differentiation of regulatory T cells (Treg) in each group were analyzed by flow cytometry. Results The morphology of PD-L1hitol-DC modified by PD-L1 gene was consistent with tol-DC characteristics, and the expression levels of CD80, CD86 and major histocompatibility complex (MHC) on the surface were low. After mixed culture with CD8+ T cells, the levels of IL-10 and transforming growth factor (TGF) -β1 in the supernatant of PD-L1hitol-DC group were higher, the levels of tumor necrosis factor (TNF) -α and IL-17A were lower, and the apoptosis of T cells and Treg differentiation were increased. Conclusions Overexpression of PD-L1 through lentiviral vectors may successfully induce the construction of bone-marrow derived PD-L1hitol-DC in DA rats, promote the secretion of anti-inflammatory factors and T cell apoptosis, induce the differentiation of Treg, and inhibit the immune response of allogeneic CD8+T cells, which provides experimental basis for the next organ transplantation immune tolerance study.
