Establishment Of Murine Cytokine-Activated Donor Lymphocyte Infusion Treatment Model And Preliminary Mechanistic Investigation Of Relevant Graft Versus Leukemia Effect

Objective：To establish the mouse model of relapsing leukemia after MHC-matchedallogeneic bone marrow transplantation （BMT）, and the relevant treatment by traditionaldonor lymphocytes infusion （tDLI） as well as cytokine-activated donor lymphocytesinfusion （aDLI）, in order to compare the differences of graft-versus-leukemia （GVL）effects induced by aDLI and tDLI and to investigate the related immunologicalmechanisms.Methods：The mouse model of relapsing leukemia after MHC-matched BMT wasestablished, with the erythroleukemia (EL9611,^H-2d) BALB/c mice (BALB/c^H-2dmice) asthe recipient and the DBA/2^H-2dmice as the donor. The BALB/c^H-2dmice were originatedfrom BALB/c mice by intravenous implantation with1×10⁶EL9611leukemia cells7daysbefore transplantation. Bone marrow cells （BMCs） and splenocytes （SCs） were harvestedfrom the donor mice before transplantation. Following8Gy total body irradiation （TBI）,BALB/c^H-2dmice were randomly separated into6groups （8mice in each group）. Group A:only5×10⁶BMCs were transplanted; Group B:3days after5×10⁶BMCs injection,recombinant interferon-α （IFN-α） was daily injected subcutaneously （20000U/d）; GroupC1:5×10⁶BMCs combined with1×10⁷SCs injection; Group C2:5×10⁶BMCscombined with2×10⁷SCs injection; Group D1: after3days of5×10⁶BMCs and1×10⁷SCs injection, recombinant IFN-α was daily injected subcutaneously （20000U/d）; GroupD2: after3days of5×10⁶BMCs and2×10⁷SCs injection, recombinant IFN-α was dailyinjected subcutaneously （20000U/d）. The survival time, leukemia burden, graft-versus-hostdiseases （GVHD） and histopathological manifestations in each group were thoroughlydetermined. Moreover, with the method of flow cytometry, peripheral blood T lymphocyte subsets （such as CD3⁺、CD4⁺、CD8⁺T lymphocytes） and the expression of CD69（activation marker） were detected in both C2and D2groups.Results：The average survival time of group A, B, C1, C2, D1and D2were （11.88±1.89）d,（14.12±1.73）d,（29.63±2.00）d,（36.00±9.91）d,（32.50±2.07）d and （46.25±11.45）d, respectively. It showed that mice in group C1, C2, D1and D2survived longerthan that of group A and B （p<0.05）. The median survival time of group D1and D2werelonger than that of group C1and C2; and the difference between D2and C2wassignificant （p<0.05）. Varying clinical manifestations concerning acute GVHD （aGVHD）,such as weight loss,posture, activity, fur texture and so on, were found in group C1, C2, D1,and D2, but not in group A and B. The clinical scores for aGVHD in group C1, C2, D1andD2were1.88±0.64,2.63±0.74,2.25±0.71,3.25±0.71, respectively. Among them, thescores of group D1and D2were significantly higher than that of C1and C2, respectively（p<0.05）. The pathological manifestations in skin, liver and intestine were consistent withthat of clinical aGVHD scores. It showed that1/8and3/8mice survived so far in group C2and D2, respectively. In group A, B, C1and D1, all mice were died for leukemia accordingto pathological evidence （peripheral blood smear and histopathology results）, but notassociated with aGVHD. Furthermore, in group D2, CD3⁺, CD3⁺CD8⁺, and CD8⁺CD69⁺Tlymphocytes in peripheral blood were significantly increased （p<0.05）, the ratio ofCD4/CD8was significantly decreased （p<0.05）, and the expression of CD69wassignificantly augmented, compared with that of group C2.Conclusion：In this study, we established the mouse model of relapsing leukemia afterMHC-matched BMT, with the BALB/c^H-2dmice as the recipient and the DBA/2^H-2dmiceas the donor. Upon this, we investigated several different regimes of adoptiveimmunotherapy and founded the cytokine-activated donor lymphocyte infusion （aDLI）treatment model. Our results indicated that:（1） aDLI treatment could induce moreprominent GVL effects so as to prolong the survival time for relapsing leukemia afterMHC-matched BMT;（2） aDLI treatment could trigger more remarkable aGVHD effects;（3） increased amplification and activation of CD8⁺T lymphocyte were tightly associatedwith the promoted GVL/GVHD effects induced by aDLI treatment.