Cooperation Of CD4~+T Cells And CD8~+T Cells And Release Of IFN-? Are Critical For Antileukemia Responses Of Recipient Mice Treated By Microtransplantation

Part I.Establishment and Identification of a Microtransplantation Model of Leukemia MouseBackground Previous studies have shown that "microtransplantation" can induce patients with acute leukemia/lymphoma to abtain continuous treatment response without implantation.However,the specific mechanism has not been clarified,so it is essential to establish a microtransplantation model of leukemia mouse to study it.Objective To establish a microtransplantation model of leukemia mouse and evaluate the occurrence of GVHD.Methods Female BALB/c mice(H-2kd/d)were recipients and male C57BL/6j mice(H-2kb/b)were donors.The recipients were intravenously inoculated by tail vein with WEHI-3 cells(myeloid leukemia cell line)(about 1 x 106/one mouse)5 days before microtransplantation.The chemotherapy with MA regimen was started 4 days before microtransplantation and stopped at-1d.The spleen mononuclear cells(sMNC,6 x 107/one mouse)were infused within 8 hours while the same amount of saline solution was given to the control group.Flow cytometry was applied to detect the chimerism rate of CD3+ cells in peripheral blood of donors 7 and 14 days after microtransplantation.The detection of bone marrow leukemia load was performed three weeks after inoculation with WEHI-3.General characteristics of the recipient mice,such as mental condition,appetite,activity,diarrhea,skin,weight and incidence of early death,were observed and recorded.The incidence of GVHD was assessed once daily after transplantation.The degree of GVHD was scored based on five indexes,namely weight loss,posture,activity,hair texture and skin integrity.Specimens of liver,spleen and small intestine were taken on day +3 after transplantation.Next,the specimens were fixed with 10%formaldehyde solution,conventionally paraffin-embedded,sectioned,hematoxylin and eosin stained and observed under a light microscope.Results Compared with control group,the leukemia load of groups of leukemia mice with microtransplantation decreased obviously.The chimerism rate was(0.12+0.03)%at +7d after transplantation and decreased to zero at +14d.No early death was observed until the end-point of the experiments.Regarding weight,mental condition,appetite,activity,diarrhea,skin and hair,no significant differences were present among the groups.No symptoms or signs including rash or skin ulcers typically associated with acute GVHD were observed.Furthermore,no significant histopathological differences in tissue biopsy specimens of intestine,liver and spleen were detected.Conclusion The microtransplantation model of leukemia mouse was successfully established and the validity and safety of the microtransplantation were preliminary proved while no obvious GVHD occurred.These facilitated the study of the mechanism of microtransplantation for the treatment of leukemia.Part ?.The Preliminary Study on the Mechanism of Microtransplantation for the Treatment of LeukemiaBackground The mechanism of microtransplantation for the treatment of leukemia has been previously speculated that HVG effect(host-versus-graft)may play an important role,and several immune cells and cytokines might participate in it,but it was still in lack of experimental studies to prove them.Objective To study the role of CD4+ T cells,CD8+ T cells,cytokine such as IFN-y and IL-4 on the anti-leukemia effect of microtransplantation.Methods The experiment included 8 groups:Group A,MA + cytarabine + normal saline;group B,MA + spleen mononuclear cells(sMNC)without mobilization;group C,MA +mobilized sMNC;g:roup D,MA + mobilized sMNC + IL-2;group E,MA + sMNC without mobilization + CD4+ T-cell depletion;group F,MA + mobilized sMNC + CD4+ T-cell depletion;group G,MA + mobilized sMNC + CD8+ T-cell depletion;group H,MA +mobilized sMNC + CD4+ T-cell depletion + CD8+ T-cell depletion.Flow cytometry was used to detect the count of infused CD4+ T cells and CD8+ T cells.Bone marrow leukemia load was observed under microscope.The levels of IFN-y and IL-4 were detected by ELISA at +7d.Then to assess the influence of infused CD4+ T cells and CD8+ T cells to the levels of IFN-y and IL-4,and the correlation of various parameters and the change of leukemia load.Results We demonstrate that IFN-? is critical to the antileukemia response in the mouse model of microtransplantation.Therapeutic efficacy was positively associated with the number of CD4+ T cells(Pearson's r=0.722).In addition,CD8+ T cells increased the release of IFN-? with assistance from CD4+ T cells.Interleukin-2(IL-2)could augment IFN-y release,partly by increasing CD4+ T cells(42.8%vs 35.6%,p<0.05).Conclusion Our study suggests that the release of IFN-? via the cooperation of CD4+ T cells and CD8+ T cells represents a crucial mechanism in the antileukemia responses of recipient leukemic mice treated with microtransplantation.During this process,the cooperation of CD4+ T cells and CD8+ T cells was shown to play a major role in the antileukemia effect.IL-2 might be developed to improve the efficacy of microtransplantation by increasing CD4+ T cells.