CD47 Engagement Impairs the Function and Maturation of Human Dendritic Cells Via the Inhibition of NF-κB Avtivity.
Authors: Yu Jing , Lin Maofang 
Published: October 2019

Abstract
OBJECTIVE Dendritic cells (DCs) are important functional antigen-presenting cells that play an essential role in initiating and modulating immune responses. Their mature states are crucial for the induction of T-cell-mediated immune reactions. Therefore, The mechanism of T cell anergy related with DCs and research in applying immature DCs to establish donor-specific immune tolerance have become one of the new frontiers in the field of transplantation immunity. The activation of nuclear factor kappa B(NF-κB) has been said to modulate the maturation of DCs, which controls the transcription of genes encoding major histocompatibility complex(MHC)antigens, and costimulator molecules for T cell activation. The CD47 Ag, a multispan transmembrane ptotein expressed on all hemopoietic cells, has an immunosuppressive function with its ligand, monoclonal antibody (mAb) or thrombospondin. In the current study, we investigate the influence of CD47 engagement by its soluble mAb B6H12 on the maturation and function of cultured DCs. Further to observe any results could be ascribed to the effect of B6H12 mAb on the nuclear translocation and DNA binding of NF-κB.
METHODS Monocyte-derived DCs were propagated in granulocyte-macrophage colony stimulating factor (GM-CSF) combined with lipopolysaccharide (LPS) and interleukin(IL)-4, in the presence or absence of soluble anti-CD47 monoclonal antibodies (anti-CD47 mAbs, B6H12). The phenotype of DCs(CD80, CD86, CD83, CD1a, HLA-DR) were detected by the flow cytometry. Semi-quantitative RT-PCR and ELISA methods were used to analyse the mRNA and protein expression levels of interleukin-12(IL-12). The antigen-presenting functions of DCs were determined in one-way mixed leukocyte reaction by Brdu-ELISA. Electrophoretic mobility shift assay (EMSA) was applied to examine the activity of NF-κB.
RESULTS Both the immature and the mature DCs express CD47, which has a ratio of 94-98%. The cell immune phenotype was lower in B6H12 mAb treated DC group than that in untreated groups. The data were as follow:CD80+(68.14±7.41)% vs (89.17±8.59)%; CD86+ (67.33±4.71)% vs (87.27±3.56)%;CD83(40.08±14.80)% vs (72.77±8.68)%;Cdla+(66.45±4.06)% vs (95.93±3.03)%; HLA-DR(40.67±13.48) vs (98.97±1.01)%, respectively. The mRNA levels of IL-12 P40 was decreased (P<0.01) with the treatment of B6H12 mAb. Pre-exposure of developing DCs to 10μg/ml B6H12 considerably reduced their subsequent ability to secret IL-12 P70 upon stimulation by LPS, comparing with the other two control groups [(36.6±3.83)pg/ml vs (80.43±8.24)pg/ml vs(83.7±9.58 )pg/ml] (P<0.05). It is also noted that the inhibition of IL-12 P70 release by B6H12 mAb was dependent on dose at the range of 2.5 - 10μg/ml, and the inhibition can be seen in DCs treated with B6H12 mAb at the dose of 2.5μg/ml. The data of the mixed leukocyte reaction were consistent with the flow cytometry results (P<0.01). Pre-exposure to B6H12 mAb during the development of DCs resulted in a dramatic decrease of the DNA binding activity toward protein in the nucleus. Moreover, this inhibition was also seemed to be dose dependent (2.5μg/ml, 5μg/ml, 10μg/ml) and the density scan values were:126.1±4.32, 103.46±10.96, 72.80±10.08.
CONCLUSION This results indicate that the CD47 ligation by B6H12 mAb exerts a negative effect on the maturation and function of in vitro cultured DCs through the inhibition of NF-κB binding activity, which suggest its favorable role in the induction of modulation of immune tolerance.