The Effect And Mechanism Of Single High-dose Radiation Therapy On Lymph Node Homing And T Cell Activation Of Dendritic Cells

Background:The clinical application of X-ray hypofractionated radiotherapy is increasing,and its immune effect is widely recognized.Dendritic cells(DCS)are one of the most important antigen-presenting cells.They are mainly distributed in peripheral tissues and play an important role in immune surveillance.When it is stimulated by antigen,it can migrate to lymphoid tissue and present antigen to the T cells.Therefore,whether and how much DC can efficiently migrate to the T cell rich region is the key step to determine its function.However,there is no consistent conclusion on whether X-ray irradiation affects the homing ability of DC.Objective:To explore the effect of single high dose X-ray irradiation on the homing ability of DC to lymphoid tissue and T cell activation,and to reveal the molecular mechanism.Methods:in order to be close to clinical practice,we used medical linear accelerator as radiation source to irradiate DC at 2,5,10,15 or 20Gy.Then,flow cytometry was used to analyze the effect of X-ray irradiation on DC mature phenotype;immunofluorescence was used to analyze the effect of irradiation on DC cytoskeleton;reactive oxygen species probe was used to detect the induction of reactive oxygen species in DC;Western blot was used to analyze the signal pathway related to cytoskeleton rearrangement;the ability of lymph node homing in vivo was detected by small animal imaging technology;the ability of T cell primming d by DC was detected by adoptive DC immunoassay.Results:within 72 hours,the survival of DC was not affected by 2-20Gy irradiation.CD40,CD80,CD86,CXCR4 and CCR7 were up-regulated in a dose-dependent manner,and the secretion of pro-inflammatory factor TNF-α was increased.In addition,compared with the 2Gy group,20Gy irradiation can significantly promote the homing of peripheral tissue DC and circulating DC to lymphoid tissue.Further studies showed that the enhancement of cytoskeleton rearrangement and the up-regulated expression of chemokine receptor on the surface of DC cells were the main factors for the enhancement of DC migration induced by high-dose irradiation.At the same time,the increase of reactive oxygen species induced by radiation and the activation of RhoA/Rock1 signaling pathway are the main mechanisms of DC cytoskeleton rearrangement.Finally,compared with the 2Gy group,DC after 20Gy irradiation has stronger T cell activation ability in vivo,which is manifested by the increase of the number and proportion of OVA257-264 specific CD8+T cells and the up regulation of CD44/CD69 and TNF-α on the surface of reactive CD8+T cells.Finally,inhibition of ROS will greatly reduce the degree of T cell activation in vivo,suggesting that the accumulation of ROS induced by single high dose radiation is the key to promote DC to activate T cells.Conclusion:single high dose irradiation has advantages in promoting DC homing to lymph nodes and T cell activation compared with conventional fractionated dose irradiation.