Establishment Of Mouse Model Of Decitabine Combined With Cytokine-activated Donor Lymphocyte Infusion For Treatment Of Acute Leukemia Relapse After Transplantation And Investigation Of Its Concerned Mechanisms

Objective: To establish four kinds of mouse models with the erythroleukemia(EL9611, H-2d) burden BALB/c mice(H-2d) as the recipient and(C57BL/6J×BALB/c) F1 mice CB6F1(H-2d/b)as the donor:(1) acute erythroleukemia relapse model after MHC haploidentical allogeneic bone marrow transplantation(BMT),(2) traditional donor lymphocytes infusion(t DLI) treatment model,(3) interferon-alpha(IFN-α) activated donor lymphocytes infusion(a DLI) treatment model, and(4) decitabine combined with IFN-α activated donor lymphocytes infusion(a DLI) treatment model. To compare the efficacy of graft versus leukemia(GVL) effects and severity of graft-versus-host disease(GVHD) between different DLI models, and to investigate related mechanisms.Methods:To establish the relapsed mouse models with the erythroleukemia(EL9611, H-2d) burden BALB/c mice(H-2d) as the recipient and the(C57BL/6J×BALB/c) F1 mice CB6F1(H-2d/b)as the donor. The BALB/c H-2d mice were implanted with 1×106 EL9611 fresh leukemia cells by intravenous injection a week before transplantation. The BABL/c(H-2d) mice were given X-ray linear accelerator total body irradiation(TBI) with a total dose of 8Gy and by a rate of 50 c Gy/min on transplant day. Bone marrow cells(BMCs) and splenocytes(SCs) were harvested from the donor mice under aseptic conditions before transplantation. Within 4-6 hours after TBI, conducting CB6F1(H-2d/b) mice �' BALB/c(H-2d)mice allogeneic bone marrow transplantation. The experiment was divided into two parts: The pre experiment was to find the appropriate spleen cell dosage, following 8Gy total body irradiation(TBI), BALB/c H-2d mice were randomized into 3 groups(10 mice in each group). Group A: only 5×106 BMCs were transplanted; Group B: 5×106 BMCs combined with 1×107 SCs(B1), 1.5×107 SCs(B2), 2×107 SCs(B3); Group C: 5×106 BMCs combined with 1×107 SCs(C1), 1.5×107 SCs(C2), 2×107 SCs(C3), recombinant IFN-α was daily injected subcutaneously(20000U/d) after 3 days. The survival time, leukemia burden, graft-versus-host diseases(GVHD) and histopathological manifestations in each group were sudied. The second part include 3 groups: Group D( t DLI group): 5×106 BMCs combined with 1.5×107 SCs were injected; Group E(a DLI group): 3 days later after 5×106 BMCs and 1.5×107 SCs were injected, recombinant human IFN-α-2b was daily injected subcutaneously(20000U/d); Group F: 3 days later after 5×106 BMCs and 1.5×107 SCs were injected, decitabine(1mg/kg/d) was injected daily for three days, and recombinant human IFN-α-2b was daily injected subcutaneously(20000U/d). The survival time, leukemia burden, graft-versus-host diseases(GVHD) and histopathological manifestations in each group were studied. By flow cytometry, activitied CD8+ T lymphocytes from peripheral blood,peripheral blood and GVHD target organs(spleen, liver, lung) CD4+CD25+Treg cell were detected in group D, E, F. By enzyme-linked immunosorbent assay(ELISA), IFN-γ、TNF-a、TGF-β、IL-10 and IL-35 expression differences in mice’s peripheral blood plasma post transplantation in three groups were studied.Results:Analysis of the data from three batches of experiments, the average survival time of group A, B1, B2, B3, C1, C2, C3 were(9.8±2.3)d,(19.0±5.12)d,(23.4±4.03) d,(26.4±4.53)d,(22.5±4.19)d,(32.9±3.78)d, and(20.6±4.59)d respectively. In group A, B1, C1 all mice died of leukemia according to pathological evidence(peripheral blood smear and histopathology results) in the early stage after transplantation, and group C2 early died of severe acute GVHD. Considering the survival time of different spleen cells infusion quantity group and experiment controllability, spleen cell dosage choosed 1.5×107. The average survival time of second part were: group D(21.7±7.76)d, group E(34.9±8.04)d, group F(45.6±8.48)d. It showed that mice in group E and group F survived longer than that of group D(p<0.05). It showed that 11/30 and 20/30 mice survived so far in group E and F respectively, all mice were died of leukemia in group D. Varying clinical manifestations concerning acute GVHD(a GVHD), such as weight loss, posture, activity, fur texture and so on, were found in group D and group F, but more severe in group E. The clinical scores for a GVHD in group D, E and F were 1.88±0.64, 1.74 ± 0.72, 3.32 ± 0.91 respectively. Among them, the scores of group E were significantly higher than that of group D and F(p<0.05). On the10 day, 17 day and 24 day post transplantation, the liver, intestine and skin of mice in three groups were collected for pathological examination. The results showed that the lymphocytic infiltration in group E was severe than group D and group F, 17 d showed the most serious performance. Flow cytometry results also showed that: at the same time post transplantation, the percentage of activation CD8+ T cell in mice’s peripheral blood of group E and group F were higher than that of group D(P<0.05). What’s more, the percentage of Treg cell in peripheral blood and GVHD target organs of group F was higher than group D and group E(P<0.05). ELISA results displayed that at the same time post transplantation, TNF-a and IFN-γ level of group E was higher than that of group D, and the differences was significantly(P<0.05). Inhibitory cytokines like TGF-β and IL-10 level of group F was significantly higher than group D and group E, and the differences was significantly(P<0.05). IL-35 level of group F was higher than other groups but with no statistical significance(P>0.05).Conclusion: This study successfully established the mouse model of acute erythroleukemia(EL9611, H-2d) relapse after MHC-haploidentical allogeneic bone marrow transplantation(BMT), with the BALB/c H-2d mice as the recipient and the CB6F1H-2d/b mice as the donor. Under the different regimens of adoptive immunotherapy, this study also established the mouse model of traditional donor lymphocyte infusion(t DLI) treatment and interferon-alpha(IFN-α) activated DLI(a DLI) treatment, decitabine combined with a DLI treatment respectively. Moreover, the preliminary study indicated that IFN-α may enhance and prolong the proliferation, activation, killing effect of CD8+CTL and Th1 cytokines to the GVL effects of DLI so as to prolong the survival time of mice, decitabine can enhance the Treg and inhibitor cytokines to reduce the incidence and severity of GVHD and did not affect GVL.