Experimental study of G-CSF alleviating graft-versus-host disease after mixed bone marrow transplantation in mice

Objective： How to reduce the incidence and severity of acute graft-versus-host disease （aGVHD） is a crucial step to improve the overall survival of allogeneic bone marrow transplantation （allo-BMT）. The low incidence of severe aGVHD observed in allogeneic peripheral blood stem cell transplantation （allo-PBSCT）, which may be related to modulating immune function of T lymphocytes by granulocyte colony-stimulating factor （G-CSF） primed donors. The study aimed to explore whether aGVHD could be alleviated by syngeneic bone marrow mixed with G-CSF-mobilized H-2 haploidentical marrow grafting. Methods： Female BALB/c mice and neonatal BALB/c mice were recipients and male （BALB/c × C57BL/6）F1（BCF1） mice were donor mice respectively. Donor mice were injected subcutaneously with G-CSF daily at 0.01 μg/g body weight or saline for 6 days, and splenocytes were harvested on day 6. Spleen index （SI） represented GVHD in neonatal mice after the intraperitoneal injection of mixed spleen cells. Lethally irradiated （^60Co, 8.5 Gy） adult mice were transplanted with a mixture of syngeneic plus G-CSF-mobilized （control diluents） H-2 haploidentical marrow cells. Survival time and survival rate of the recipients were observed after mixed marrow transplantation （MBMT）. GVHD was assessed by observing signs of weight loss, ruffled fur, diarrhea and histological change of skin, liver and small intestines. Enzyme-linked immunosorbent assay （ELISA） method was used to detect cytokines （IL-2, IL-4 and INF-γ）. Fluorescence-activated cell sorting （FACS） analysis was used to detect T cells phenotype. Results： （1） The neonatal mice subject to injection of 2：1 and 1：1 mixed spleen cells and H-2 haploidentical spleen cells all suffered from aGVHD. The severity of aGVHD in recipient mice receiving G-CSF-mobilized splenocytes was dramatically reduced. （2） The aGVHD signs and histological change were observed in most mice of 2：1 and 1：1 MBMT groups. However, the survival time of G-CSF-mobilized MBMT was longer than in control groups and these mice had signs of moderate GVHD. （3） L3T4^＋ cells and relative ratio in both subsets was significantly reduced in G-CSF-treated donor mice. The total number of Thyl.2 and lyt2^＋ cells was increased after G-CSF pretreatment of donors, but no statistical difference. （4） The supernatants from a primary MLR were collected at 48 h for cytokine measurement. The results showed an increased production of IL-4 and a decreased production of IL-2 and INF-γ after stimulating with concanavalin A for 48 h. Conclusion： The GVHD could be reduced using syngeneic bone marrow mixed with H-2 haploidentical marrow cells. The severity of aGVHD in recipient mice receiving G-CSF-mobilized splenocytes or marrow cells could be further moderated, which is associated with increased IL-4 production and decreased IL-2 and INF-y production.

Induction of graft-versus-leukemia effect using a mixture of syngeneic plus G-CSF primed haploidentical bone marrow grafts in mice

Objective： To explore whether the graft-versus-leukemia （GVL） effects could be enhanced and acute graft-versus-host disease （aGVHD） could be relieved by syngeneic bone marrow mixed with G-CSF-primed H-2 haploidentical marrow grafting. Methods： Female L615 （H-2^k） mice were recipient mice and male （615×BALB/c） F1 （6BF1） （H-2^k×H-2^d） mice were donors respectively. Donor mice were injected subcutaneously with G-CSF daily at 0.01 μg/g for 6 days, and splenocytes were harvested on day 7. A total of 615 mice were loaded with L615 tumor cells and received 8.5 Gy （^60Co γ-ray） irradiation three days later, followed by a mixed bone marrow transplantation （MBMT）. The allo-grafts consisted of a mixture of syngenetic plus G-CSF-mobilized （control diluents） H-2 haploidetical marrow cells. GVL effects were monitored by survival time and survival rate of recipient mice. GVHD was assessed by clinical signs of weight loss, ruffled fur, diarrhea and histological changes of skin, liver and small intestines. AIIogeneic chimerism was detected using cytogenetic analysis. Enzyme-linked immunosorbent assay （ELISA） method was used to detect cytokines （IL-2, IL-4 and IFN-γ）. Fluorescence-activated cell sorting （FACS） analysis was used to detect T-cell phenotype. Results： （1） The mice merely received L615 leukemia cells were all died of leukemia. The L6t5 mice of 3：1 and 4：1 MBMT groups survived longer than those post syngeneic BMT （P 〈 0.01）. （2） The survival time of mice in the G-CSF-treated MBMT groups was longer than that of non-primed MBMT groups （P 〈 0.05）. Administration of G-CSF-treated 6BF1 mice could markedly increase the survival rate of 3：1 and 4：1 MBMT mice （P 〈 0.01） with little or a little GVHD. （3） As the post-transplanted time prolonged, the rates of allogeneic chimerism were decreased. The chimerism rates decreased to zero when the mice died of leukemia relapse. （4） L3T4^＋ cells and relative ratio in both subsets were significantly reduced in G-CSF-treated donor mice. After G-CSF-treated donors, splenocytes from recipients at day 14 post-MBMT showed an increased production of IL-4 and a decreased production of IL-2 and IFN-γ. Conclusion： Syngeneic bone marrow mixed with H-2 haploidentical marrow grafts had a potential way to increase GVL effects, and this GVL effects could be enhanced with little or a little GVHD by G-CSF pretreatment of donors. Improved survival in recipients of G-CSF-mobilized donors is associated with increased IL-4 production and decreased IL-2 and IFN-γ production.