Effect Of X-ray Irradiated Lymphoma Cells Combinated With RhIL-15 On Allo-PBMCs Cytotoxicity

Background and ObjiectivesMalignant lymphoma is a group of malignancies of the lymphatic system from the blood, the incidence rate is about 5/10 million people, recent years the incidence is rising. Hodgkin's lymphoma get better treatment of combination chemotherapy, combined with radiotherapy. NHL treated with combination chemotherapy, but conventional treatment easily relapse, salvage treatment is poor, and the toxic effect of chemotherapy drugs is harmful to the bodies.The biological immunity treatment was considered a accurate effect method to the tumor which is after the surgery, radiotherapy and chemotherapy. Current immunotherapy including interferon, cytokines, monoclonal antibodies, tumor vaccines, immune gene therapy and adoptive immunotherapy. Adoptive immunotherapy (ATI) transmitte immune cells to cancer patients with the effect of anti-tumor directly or stimulating the body to againt tumor. In addition to CIK cells, NK cells are considered ideal immune effector cells.T cells and NK cells as the main immune cells killing the tumor cells, particularly the central part of tumor immunotherapy is that T cells recognize the tumor antigen and then kill the tumor cells specifically. The two conditions of immune cells Anti-tumor include:tumor cells can activate immune cells, activated immune cells can kill tumor cells. Present problems are following:insufficient activation of immune cells and tumor cells are resistance to immune cells.Recent studies show that radiation, anthracycline chemotherapy can induce early apoptosis in tumor cells. Then the calreticulin (CRT) translocate from the cytoplasm to the membrane, recognited and phagocytosis by the surface receptor of dendritic cells, and then the tumor antigen are presented to T cells, induce the anti-tumor immune response.This sdudy was to detect the effect of X-ray irradiated lymphoma cells in combination with rhIL-15 on Allo-PBMCs cytotoxicity.MethodsPart I Induction of apoptosis of human Burkitt lymphoma cell lines Raji by X-ray radiationHuman Burkkit lymphoma cell lines Raji was inducted by X-ray radiation, the dosage was 0,4,6,8,16Gy respectively, OGy was normal control group. Raji cells were cultured in the incubator after radiation, at 12,24,48h cells were collected for testing. Phasecontrast mcroscope was used to observe the cells morphological change, flow cytometry was used to detect the changes of apoptosis, cell cycle. Method of methyl cellulose semi-solid culture was used to observe the colony formation of Raji cells after irradiation.Part II Effect of X-ray irradiated lymphoma cells in combination with rhIL-15 on Allo-PBMCs cytotoxicityPeripheral blood mononuclear cells separated from 50ml Peripheral blood of healthy volunteers. Collecting Raji cells after 8Gy irradiation at 48h, adding to PBMC cells suspension at ratio of 1:20. Set four groups:control group (PBMCs), rhIL-15+PBMCs group, irradiated Raji cells+PBMCs group, rhIL15+irradiated Raji cells+PBMCs group, Cultured for 14 days, the characteristics including Secretion of INF-y, cytotoxin, cell clone, phenotype of NKG2D were analyzed.Statistical methods:The results of experimental data with mean±standard deviation (±s) that significant for the 0.05 standard, using SPSS13.0 statistical packages for statistical analysis. Using two-sample t-test, Heterogeneity of variance used satterthwaite approximate t-test; Comparisons of means among groups using one-way ANOVA; inter-group comparison was significant homogeneity using LSD method, with variance arrhythmia Dunnett's T3.ResultsPart I Induction of apoptosis of human Burkkit lymphoma cell lines Raji by X-ray radiation①Trypan blue staining results showed that, by comparing the living cells and inhibition rates between irradiated Raji cells and control cells at 12,24,48h, found that the number of irradiated Raji cells no longer increase. To detect the number of living cells with different irradiation doses (0,4,8,12,16Gy) at different time points (12,24,48h):the number of living cells with different irradiation doses at 24,48h was significantly different (F=51.138,P=0.000, F=322.936,P=0.000), at the same time,with irradiation doses, the number of living cells was gradually reduced; To compare the number of living cells of each dose group at different time points:the number of living cells of each irradiation dose at different time points was significantly different (P=0.000,P=0.094,P=0.007,P=0.000), at the same irradiation dose, with time, the number of living cells was gradually reduced; To compare the inhibition rates with different irradiation doses (4,8,12,16Gy,0Gy as control group) at different time points (12,24,48h):the inhibition rates with different irradiation doses at 24,48h were significantly different(P=0.003,P=0.001), at the same time, with irradiation doses, the inhibition rates were gradually enhanced; To compare the inhibition rates of each dose group at different time points:the inhibition rates of each irradiation dose at different time points was significantly different (P=0.000,P=0.001,P=0.001,P=0.023), at the same irradiation dose, with time, the inhibition rates was gradually enhanced; X-ray obviously inhibited the growth of Raji cells in a time-and dose-dependent manner.②Cell morphology was observed under the microscope, Raji cells in nomorl control group grow agglomeratly, had uniform size, clear edge, and were bright. Some irradiated Raji cells were shrunken, bodies were larger or smaller, less refraction, and debris increased; Compared to control garoup, cells in 8Gy group were shrunken, bodies were smaller, less refraction, and debris increased; cells in 16Gy group were more significant.③Flow cytometry was used to detect the changes of apoptosis. The early apoptosis rates with different irradiation doses at 12,24h were no significant difference (P >0.05); the early apoptosis rates with different irradiation doses at 48h were significantly different (F=28.408,P=0.000),after the SNK multiple comparison, the early apoptosis rates with irradiation doses of 4,8,12,16Gy were significantly higher than OGy, the early apoptosis rate with 8Gy at 48h was (42.04±3.62) %, which significantly higher than others; The late apoptosis rates with different irradiation doses at 24,48h were significantly different (F=14.338,P=0.000, F=22.986,P=0.000); After the SNK multiple comparison, the late apoptosis rates with irradiation doses of 8,12,16Gy at 48h were significantly higher than other time points, the late apoptosis rate with 8Gy,16Gy at 24h were significantly higher than other doses; The result indicated X-ray could induce apoptosis of Raji cells. ④Flow cytometry was used to detect the changes of cell cycle. At 24h with 0,8,12 Gy irradiation, the cell percentage in different phase of cell cycle with different doses were significantly different (F=11.875,P=0.008,F=22.752,P=0.000, F=60.899,P=0.000), after the SNK multiple comparison, the cell percentage in G2/M phase with 8,12Gy were (57.86±3.31)%, (45.57±2.86)% respectively, obviously higher than OGy, the cell percentage in G2/M phase between 8,12Gy groups were no significant difference (P>0.05); In S phase, the cell percentage with 8Gy was (9.25±2.32)%, obviously less than OGy. Cell cycle of Raji could arrested in G2/M phase after X-ray irradiation.⑤In vitro colony assasy indicated, the number of colony between 0,8Gy groups at day 7,14 were significantly different (F=5.754,P=0.005,F=12.636,P=0.000) X-ray could inhibit the growth of Raji cells, also the cancer stem cells.Part II Effect of X-ray irradiated lymphoma cells in combination with rhIL-15 on Allo-PBMCs cytotoxicity①To study the E:T ratio of the Raji cells cultured with allo-PBMCs, set 6 groups: normal control (PBMCs), irradiated Raji cells:PBMCs 1:1,1:5,1:10,1:20,1:40, at day 3,cell morphology was observed under the microscope,in 1:1,1:5,1:10(E:T) groups, most PBMCs bodies were larger, cells wall were incomplete, less refraction, and cell debris increased; Control group cells were still suspended growth, cells state were good; In 1:20,1:40 group, PBMCs grow agglomeratly, some irradiated Raji cells existed; At day 6-9, in 1:1,1:5,1:10(E:T) groups most PBMCs died; In 1:20,1:40 group, PBMCs bodies and agglomerates were bigger, less irradiated Raji cells existed; At day 12-14, In 1:20,1:40 group, irradiated Raji cells disappeared. At day 14, the number of PBMCs in each groups were significantly different (F=10.602,P=0.000), after the SNK multiple comparison, the number of PBMCs in control,1:20,1:40 groups were no significant difference (P>0.05)②To observe the effect on allo-PBMCs growth and proliferation after co-culturing with irradiation cells and rhIL-15 on day 14 by counting the number of allo-PB-MCs. The numbers were significantly different (F=30.176, P<0.001), after the SNK multiple comparison, rhIL-15+PBMCs group, rhIL-15+irradiated Raji cells+PBMCs group were higher than others (P<0.05), and rhIL-15+PBMCs group was also higher than rhIL-15+irradiated Raji cells+PBMCs group (P<0.05)③At day 3 of cultured system, IFN-γwere detected by ELISA kit, secretion of IFN-y in groups were significantly different (F=17.803,P=0.001), after the SNK multiple comparison, IFN-γconcentration in rhIL-15+PBMCs group, rhIL-15+ irradiated Raji cells+PBMCs group were higher than control group, irradiated Raji cells+PBMCs group (P<0.05)④Allo-PBMCs co-cultrued with rhIL-15, irradiated Raji cells, then measured the cytocixity of allo-PBMCs againsting Raji cells, K562 cells, KGla cells. At E:T 10:1,20:1, the cytocixity of allo-PBMCs againsting K562 cells in rhIL-15+PBMCs group were (45.38±6.95)%, (73.97±5.61)%, higher than normol control group (18.93±7.25)%, (27.93±4.52)%, this indicated that the cytocixity of allo-PBMCs was enhanced co-cultrued with rhIL-15. At ratio of 10:1(E:T), the cytocixity of PBMCs againsting Raji cells in each group were no significantly difference (P>0.05), after the SNK multiple comparison, the cytocixity of PBMCs againsting Raji cells in rhIL-15+irradiated Raji cells+PBMCs group (28.97±4.17)% was higher than control group (P<0.05), but compared to KG1a cells, there were no significant differentce (P>0.05). The result indicated that rhIL-15 combinated with irradiated Raji cells could enhance the cytocixity of allo-PBMCs to Raji cells. The cytocixity of allo-PBMCs againsting three cells in rhIL-15+irradiated Raji cells+PBMCs group were significantly different (F=10.375,P=0.011), after the SNK multiple comparison, againsting Raji cells was higher than KG1a cells (P<0.05). The result indicated that allo-PBMCs co-cultrued with rhIL-15 and irradiated Raji cells could kill Raji cells specially.At ratio of 20:1 (E:T), the cytocixity of allo-PBMCs againsting Raji cells in rhIL-15+irradiated Raji cells+PBMCs group and rhIL-15+PBMCs group were higher than others (P<0.05),and rhIL15+irradiated Raji cells+PBMCs group was higher than rhIL15+PBMCs group (P<0.05); RhIL-15+irradiated Raji cells+PBMCs group agansting Raji,K562 cells were higher than KGla cells (P<0.05). The results indicate that:rhIL-15 could enhance the cytocixity of allo-PBMCs to tumor cells. irradiated cells combinated with rhIL-15 could enhance the cytocixity of allo-PBMCs to Raji cells, compaired to KGla cells, could enhance the specific cytocixity of allo-PBMCs to Raji cells.⑤Observe the effect on Raji cells colony formation in vitro after allo-PBMCs be cultured with Raji cells. There were significantly different between groups of different effector cells(F=20.681,P<0.001). After the SNK multiple comparison, clony number of untreated Raji group was significantly higher than other groups, rhIL-15+irradiated Raji cell+PBMCs group was significantly lower than other groups (P<0.05); At the same time, the clony number of rhIL-15+PBMCs group and rhIL-15+irradiated Raji cells+PBMCs group also lower than normal control group(P<0.05). However, no significant difference between this two groups (P>0.05). Allo-PBMCs co-cultrued with rhIL-15 and irradiated Raji cells can inhibit the Raji cells clones formation, but compared with rhIL-15 group, did not significantly improve the inhibition on the Raji cell colony formation.⑥The expression of NKG2D of PBMCs in groups were significantly different (P<0.05), rhIL-15+PBMCs group was higher than others, there no different between normal control and irradiated Raji cells+PBMCs groups (P>0.05)Conclusions1. X-ray irradiation can inhibite the proliferation and induce the apoptosis of Raji cells obviously; Raji cells early apoptosis rate was (42.04±3.62)% and cell cycle arrested in G2/M phase after 8Gy irradiation; X-ray did not inhibit the growth of Raji cells completely.2. We can choose irradiated Raji cells with 8 Gy after 48h culturing as activation of tumor antigen through the detection of early apoptosis rate.3. RhIL-15 can induce the expression of NKG2D of immune cells, and enhance allo-PBMCs'cytocixity to K562,Raji,KGla cells. Allo-PBMCs co-cultrued with rhIL-15 and irradiated Raji cells could enhance allo-PBMCs'cytocixity to K562, Raji cells, compared with KG1a cells, may enhance the specific cytocixity to Raji cells. Allo-PBMCs co-cultrued with rhIL-15 and irradiated Raji cells can inhibit the Raji cells clones formation, but compared with rhIL-15 group, did not significantly improve the inhibition on the Raji cell colony formation.4. Cytokine rhIL-15 can promote IFN-y secretiom of PBMCs, and induce NKG2D receptor expression, the irradiated cells in combination with rhIL-15 did not significantly improve IFN-y secretion of PBMCs,and NKG2D receptor expression has decreased.The innovation of this study:1. In this study, we explore the best dose of irradiation and culture time as the early apoptosis rate of Raji cells achieving to the highest value. After the irradiation of 8Gy dose, the growth and proliferation did not be inhibited.2. Allo-PBMCs co-cultrued with rhIL-15 and irradiated Raji cells could increase the cytocixity againsting Raji cells could be enhanced correspondingly, which can provide a new idea for the lymphoma Treatment.The value of this study:With X-ray irradiation of 8Gy, Raji cells early apoptosis rate was (42.04±3.62)%, but it did not completely inhibit the growth and proliferation of Raji cells. Allo-PBMCs co-cultrued with rhIL-15 and irradiated Raji cells could increase the specific cytotoxicity of allo-PBMCs to Raji cells, which finding a new method of cell therapy.