Study On The Changes Of Lymphocytes And Their Subpopulations In Peripheral Blood From The Patients With Carcinoma After Intensity-Modulated Radiotherapy

Objective:Radiotherapy（RT）is considered as one important treatment for the patients with cancer in advanced stage,also become one supplementary treatment for resection of tumors in early stage.It can promote the increasing survival rate and the extension of survival time of patients.However,radiotherapy may produce the adverse influence for normal tissues and cells concomitant with generation of the lethal effect of tumor cells.This method can induce the death of partial lymphocytes and their redistribution because of the high sensitivity of lymphocyte to ionizing radiation,which immunological system in human body is further modified.To the development of radiotherapeutic technology,intensity-modulated radiotherapy（IMRT）has extensively utilized to treat cancers of head-neck,prostate,chest and so on,which the method shows the higher radiation dose in target region and less damage to normal tissue.The aim of this study is to realize the influence of IMRT on the immunological function of the patients with various cancers through investigating the changes of lymphocytes and their subpopulations in peripheral blood from 183 cases prior and post radiotherapy,including lung,nasopharynx,esophagus and rectal cancer.Subsequently,the correlation of those changes and the parameters from these cases is further analyzed,which these parameters contain gender,age,stage and type of cancer.Based on present study,the experimental data will be valuable to radio-protection for immunological system in the process of IMRT and immunological supportive treatment in early period of post-radiotherapy.Methods:183 cases were collected from the resident patients with four common cancers,including lung,nasopharynx,esophagus and rectal cancer,who received IMRT at Radiation Oncology Division,West Brach of Anhui Provincial Hospital from October2015 to December 2017,of which 117 and 66 blood samples were utilized for analyses of regulatory T(T_reg)cells and immunologic function respectively.The blood samples were obtained from these patients on an empty stomach in the morning prior and post IMRTonemonthrespectively.Twomilliliteranti-freezingsamplesby ethylenediaminetetraacetic acid（EDTA）in less than 4 hours out of body were stored at room temperature.Various antibodies were used in this experiment,contained mouse anti-human monoclonal antibody of CD45,CD3,CD16,CD56,CD19,CD8,CD4,CD25,CD127.Sixty micro-liter mixture of CD45,CD3,CD16,CD56,CD19,CD8,CD4,CD25antibody and five micro-liter CD127 were added into one hundred micro-liter blood samples,followed by avoiding light and incubation for 30 min.One milliliter red cell lysis buffer was then added to complete the lysis of red cells for 5 min.After centrifuge,the samples were washed by PBS solution repeatedly.Sixty-six samples were detected for the observation of T cells and their subpopuations and one hundred seventeen samples were assayed for the investigation of regulatory T cells by flow cytometry（FCM）.The alteration of T cells and their subpopuations,B,NK and Treg cells prior and post IMRT was also observed in above sixty-six samples.Relationship of the changes of lymphocytes and various parameters was further analyzed.Two milliliter blood samples from vein were diluted by PBS solution or saline,and then were added into three milliliter separating buffer.Total blood cells were layered after gradient centrifuge in 1780 rpm/min.After lymphocytes in middle layer were drawn and counted,density of cells was regulated to 1×10⁶/L.The mixture of various mouse anti-human monoclonal antibodies,including CD45,Lin（CD3/14/19/20/56）,CD117,CD127,CD161 and CD294,was added into cells suspension to stain lymphocytes for 30 min at four degree centigrade avoiding light.After those cells were washed cell staining buffer,they were fixed paraformaldehyde for 30 min.Nine samples for the subpopulations of innate lymphoid cells（ILCs）prior and post IMRT were analyzed by FCM test,which contained ILC1,ILC2 and ILC3.Results:Firstly,the mean values of CD3⁺T,CD3⁺CD4⁺Th,CD4⁺CD25⁺,CD4⁺CD25⁺CD 127^low/-T_reg,CD4⁺/CD8⁺,CD3^-CD19⁺B cells showed the significant reduction in the patients received IMRT（Compared to prior IMRT,P<0.05,<0.01,<0.01,<0.05,<0.01,<0.01 respectively）.Conversely,CD3⁺CD8⁺Ts/Tc and CD3^-CD16⁺CD56⁺NK cells were increased in the patients post-IMRT（Compared to prior IMRT,P<0.01,<0.01 respectively）.Secondly,The results from statistical analysis of the changes of lymphocytes and various parameters displayed that gender,age and type of cancer didn’t lead to the alteration of CD4⁺CD25⁺CD127^low/-T_regeg cells and the ratio of CD4⁺/CD8⁺.However,there was one higher percentage of CD3^-CD16⁺CD56⁺NK cells in male patients compared with female patients prior and post IMRT（P<0.05）.Female patients had one higher percentage of CD3⁺CD4⁺Th cells in post IMRT（P<0.05）,while there wasn’t the statistical difference between female and male prior IMRT（P>0.05）.Although the percentage of CD3^-CD19⁺B cells in female patients was significantly higher than that of male patients before received IMRT,there wasn’t the statistical difference between female and male post IMRT（P>0.05）.While the percentage of CD3⁺T,CD3⁺CD8⁺Ts/Tc and CD3^-CD16⁺CD56⁺NK cells showed statistical difference between different diseases after IMRT,（P<0.05）there was no significant alteration between different diseases before IMRT（P>0.05）.Although the percentage of CD3^-CD19⁺B cells in different age groups wasn’t significantly difference before received IMRT（P>0.05）,there was the statistical difference in different age groups post IMRT（P<0.05）.Thirdly,the CD3⁺T,CD3⁺CD8⁺Ts/Tc and CD3^-CD16⁺CD56⁺NK cells has significant correlation with disease post IMRT（P<0.05）,in addition,the CD3⁺T and CD3^-CD16⁺CD56⁺NK cells showed significant correlation with sex post IMRT（P<0.05）,and CD3^-CD19⁺B cells has significant correlation with age post IMRT（P<0.05）.Fourthly,The percentage of ILC2 subpopulation was obviously increased,and ILC3 subpopulation decreased in the patients received IMRT（P<0.05）.Although the percentages of ILC1 subpopulation showed a litter of increasing in the patients post IMRT respectively,there wasn’t the statistical difference between prior IMRT and post IMRT（P>0.05）.Conclusion:Taken together,IMRT can induce alteration of the counts of lymphocytes and their subpopulations in the peripheral blood of patients with cancer,further influencing immunological function,and this modification of immune can’t be recovered in short-term after finished IMRT.It is therefore necessary that immunological modulating treatment in early stage should be used to promote the recovery of immunological homeostasis and protect severe inflammation and secondary cancer by radio-induced.