Establishment Of Mouse Model Of Acute Leukemia Relapse After Transplantation Treatment By Decitabine Combined With Interferon-Activated Donor Lymphocyte Infusion And Investigation Of Its Mechanisms

Objective: To establish four kinds of mouse models with the erythroleukemia(EL9611) burdening CB6F1H-2d/b mice as the recipients and C57BL/6H-2b as the donors:(1)an acute erythroleukemia relapsed model after MHC haploidentical allogeneic bone marrow transplantation(BMT),(2) traditional donor lymphocyte infusion(t DLI) treatment model,(3) interferon-alpha(IFN-α) activated DLI(a DLI) treatment model,(4)decitabine(DAC) combined with a DLI(DAC+a DLI) treatment model. To compare the efficacy of graft versus leukemia(GVL) and severity of graft-versus-host disease(GVHD)between different DLI models, as well as to investigate related mechanisms.Methods : The relapsed mouse model after haploidentical allogeneic BMT was established by using CB6F1H-2d/b mouse which is the(C57BL/6×BALB/c) F1 mouse as the recipient, and C57BL/6H-2b mouse as the donor. Bone marrow cells(BMCs), splenocytes(SCs) cells from C57BL/6 mice and fresh EL9611 cells from BABL/c mice were prepared before transplantation. The CB6F1 mice were given total body irradiation(TBI) with11.5Gy using an X-ray source, within 4-6 hours, assigned to BMC, DLI or EL9611 cells as demands of different models. The experiment was divided into two parts following TBI:The pre-experiment was to establish different disposal model(group A) and to explore the appropriate spleen cell dosage(group B and C). Group A1: no more disposal; Group A2(The relaped model): 2×106 EL9611 cells and 5×106 BMCs were given; Group A3: 2× 106 EL9611 cells, 5 × 106 BMCs and 3 days later, recombinant human IFN-α-2b(rh IFN-α-2)(2 × 104U/d) was daily injected subcutaneously; Group B: 2 × 106 EL9611 cells, 5×106 BMCs, at the same time, 1×107 SCs(B1), 1.5×107 SCs(B2), 2×107 SCs(B3)were given; Group C: 2×106 EL9611 cells, 5×106 BMCs and 1×107 SCs(C1), 1.5×107SCs(C2), 2×107 SCs(C3) were given, rh IFN-α-2b(2×104U/d) was daily injected after 3days until severe GVHD occurred. To make sure the best spleen cell dosage, the survival time, GVL effect and GVHD states in each group were studied. Then, the second part include 3 groups after TBI(Group D、E、F). Group D( t DLI group): 2×106 EL9611 cells,5×106 BMCs combined with 1.5×107 SCs were injected; Group E(a DLI group): 2×106EL9611 cells, 5 × 106 BMCs and 1.5 × 107 SCs were injected, and 3 days later,rh IFN-α-2b(2×104U/d) was daily injected subcutaneously; Group F: 3 days later after 2×106 EL9611 cells, 5×106 BMCs and 1.5×107 SCs were injected, DAC(1mg/kg/d) was injected daily for three days, and rh IFN-α-2b(2×104U/d) was daily given. The survival time, leukemia burden, GVHD and histopathological manifestations in each group were observed. By flow cytometry, activitied CD8+T lymphocytes of peripheral blood and Treg cells from peripheral blood and GVHD target organs(liver, spleen, lung) were detected in group D, E, F. By enzyme-linked immunosorbent assay(ELISA), TNF-a and TGF-βexpressions in mice’s peripheral plasma post transplantation in three groups were detected.Results:In the pre-experiment, the median survival time of group A2, A3, B1, B2, B3,C1, C2, C3 were(8.4±0.9)d,(9.5±1.0)d,(20.4±1.8)d,(22.6±1.4)d,(30.4±2.5)d,(22.9±1.5)d,(34.7±2.4)d,(19.5±1.7)d respectively. The mice of group A1 died early after TBI, because of hematopoiesis failure. In group A2, A3, B1, C1, all mice died of relapse according to pathological evidence as peripheral blood smear and histopathology results,studied in the early stage after allogeneic BMT. And most mice of group B3、C3 were early died of severe acute GVHD considering that too many SCs were given. To sum up group B and C, spleen cell dosage choosed 1.5×107. The median survival time of second part were: group D(t DLI)(22.6±1.4)d, group E(a DLI)(34.7±2.4)d, group F(DAC+a DLI)(39.1±2.9)d. It showed that P(a DLI vs t DLI)=0,P(DAC+a DLI vs a DLI)=0.01,P(DAC+a DLI vs t DLI)=0. It was observed that while all mice were died of leukemia in group D, 10/30 and 20/30 mice survived more than 45 days after DLI treatments in group E and F respectively. The clinical scores for acute GVHD in group D, E and F were2.5±1.2, 3.7±1.4, 2.2±1.0, respectively. Meanwhile, the scores of group E were significantly higher than group D and F, P(a DLI vs t DLI)=0.001,P(DAC+a DLI vs a DLI)=0.000.On the 14 day, 21 day and 28 day post transplantation, the livers, intestines and skins of mice in three groups were collected for pathological examination in order to study the GVHD in target organs. The results showed that there were more of lymphocytic infiltration in group E than group D and group F, the severest performance was presented at 21 day. Flow cytometry results showed that: the percentages of activated CD8+ T cell in peripheral blood of group E and group F, at the same time post transplantation, were higher than that of group D. However, of group F, the percentages of Treg cells in peripheral blood and GVHD target organs was higher than that of group D and E. ELISA displayed that, at the same time, TNF-a levels of group E and F were higher than that of group D.While inhibitory cytokines TGF-β level of group F was higher than group D and E.Conclusion: This study established the mouse model of acute erythroleukemia relapsed after MHC haploidentical allogeneic BMT, with the CB6F1H-2d/b mice as recipients and the C57BL/6H-2b mice as donors. On this basis, this study also established 3different models of t DLI treatment, a DLI treatment and DAC+a DLI treatment, respectively.Moreover, the study indicated that IFN-α-2b may enhance and prolong the proliferation,killing effect of CD8+CTL and cytokines to the GVL effects of a DLI so as to prolong the survival time of mice, DAC can enhance the Treg and inhibitor cytokines to reduce the incidence and severity of GVHD and did not sacrifice the effect of GVL.