Experimental Study On The Role Of Alloreactive NK Cells In Lung Cancer Treatment With Haploidentical Bone Marrow Transplantation In Mice

Part One:The role of NK cells in the nonmyeloablative bone marrow transplantationObjects:To observe the roles of alloreactive NK(allo-NK) cells in nonmyeloablative haploidentical bone marrow transplantation.Methods:We used C57BL/6J female mice(H-2D^b) as recipients and C57BL/6J×BALB/c F1 female(H-2D^b/d) as donor mice.We used fludarabine and cyclophosphamide as the nonmyeloablative conditioning regimen.The NK cell subset was isolated by magnetic beads separation columns.The purity of NK cells is detected by flowcytometry.Cytotoxicity effect of purified NK cells subset against Yac-1 lymphoma cell line and auto-lymphocytes and allo-lymphocytes were measured using LDH-release assay.The recipient mice were divided into four groups:group A,B,C and D.The mice in group A received the conditioning chemotherapy regimen without bone marrow transplantation.The mice in group B received the conditioning chemotherapy and donor NK cell infusion without transplantation.The mice in group C received the same chemotherapy regimen as group A and were given bone marrow transplantation.The mice in group D received chemotherapy and donor NK cells as conditioning regimen and then were given bone marrow transplantation.We counted the mononuclear cells of the spleen and bone marrow of the recipient mice in group A and B to observe the myeloablative effect of NK cells.We observed the intensity of GVHD after transplantation, including the weight loss,the inactivity,piloerection and the pathological changes of the liver,the kidney and the bowel in group C and D;We detected the chimerism through detecting the proportion of H-2D^d cells by flowcytometry after bone marrow transplantation;In one-way mixed lymphocyte culture,lymphocytes from F1 generation mouse were collected and treated by mitomycin as the stimulating cells and the lymphocytes from the transplanted mice as responsing cells;Results:The purity of NK cells in spleen was(74.63±5.5)%after isolation compared to(19.85±4.27)%before isolation.The cytotoxic activity of NK cells from donor F1 mice was (65.52±8.76)%against Yac-1 cells and(60.18±1.03)%against lymphocytes from the recipient C57BL/6J mice.The NK cells from the recipients had no cytotoxic effect against auto-lymphocytes.The myeloablative effect of mice in group B was better than that in group A.But there was no significant difference in the number of spleen mononuclear cells between the two groups.The proportion of H-2D^d cells of the mice in group D was significantly higher than that in group C.The proportion of donor cells in spleen was(7.50±0.70)%in group C versus(46.20±5.00)%.The proportion of donor cells in bone marrow was(10.2±2.40)%versus(28.7±5.9)%in group D.The result of mixed lymphocyte culture demonstrated that the mouse lymphocytes in group C and D were both less proliferated compared to the normal C57 mice.The lymphocytes in group B were less proliferated than that in group A.Conclusion: Alloreactive NK cells can help to ablate the donor hematopoietic cells,promote engraftment in the haploidentical nonmyeloablative bone marrow transplantation without obvious GVHD and induce the recipient tolerance of donor lymphocytes.Part 2:The anti-tumor effect of DLI after MHC haploidentical bone marrow transplantation and the in vivo distribution of DLIObjects:Establish the animal model of tumor recurrence after bone marrow transplantation and observe the anti-tumor effect and distribution of DLI.Methods: Bone marrow transplantation and the isolation of NK cells were performed as described in Part 1.We measured the cytotoxic effect of the donor purified NK cells subset against LLC cancer cells using LDH releasing assay in vitro.First,we establish the tumor recurrence modol after transplantation.Fourteen days after transplantation,the mice were inoculated LLC lung cancer cell line subcutaneously,1×10⁶ cells per mouse.Ten days after inoculation,which was the time to start the treatment of DLI,the tumor masses emerged subcutaneously.We collected lymphocytes from F1 mouse and infused the cells into the recipients.Every mouse was received 2×10⁷ cells.Second,we detected the distribution of donor lymphocytes after DLI.The recipient mice were divided into two groups according to the conditioning regimens:the chemotherapy group and the chemotherapy plus NK cell group.Eighteen days after transplantation,the spleen cells collected from F1 mouse, were marked with the isotope ³²P-ATP.The donor lymphocytes were then infused into the recipients.At different time after infusion,we detected the radioactivity of peripheral blood,lung,liver,spleen,kidney,bowel and bone marrow.Third,we observed the anti-tumor effect of DLI.The recipients were divided into five groups: group A,B,C,D and E.The mice in group A were given chemotherapy as the conditioning regimen before bone marrow transplantation without DLI after transplantation;the mice in group B were given chemotherapy and donor NK cells as the conditioning regimen without DLI;the mice in group C were given chemotherapy as conditioning and with the treatment of DLI;the mice in group D were given the same conditioning as group B but with DLI;the mice in group E were not given transplantation and DLI.Fourteen days after transplantation,we inoculated Lewis lung cancer cells subcutaneously in all the mice.Twenty fourdays after transplantation,we infused the donor lymphocytes to the recipients and again on the twenty eighteenth day.We measured the volume of the tumor.Results:The cytotoxic effect of donor NK cells against the LLC tumor cells in vitro was(65.52±8.76)%.The radioactivity in lung,spleen,kidney and liver was significantly higher in the recipient mice of the chemotherapy plus NK cell group than that of the chemotherapy group after DLI.Before the treatment of DLI,there was difference in tumor size between group A and B as well as between group B and E.After DLI,the tumor size of mice in group E was significantly larger than that in other groups.The tumor size in group D was smaller than that in all other groups.The tumor size in group D was significantly smaller than that in group C.Conclusion:The accumulative concentration of lymphocytes from DLI after transplantation is significant higher in the chemotherapy plus NK cell group than that in the chemotherapy group.DLI after transplantation has the therapeutic potential against recurrence of Lewis lung cancer.