The Study Of The Impact Of CXCR4 Blockade On Graft-versus-leukemia Effects And Graft-versus-host Disease

Background and Objectives:Acute leukemia is a group of hematological malignancies that severely threaten the health of human beings.Chemotherapy is still the backbone for the treatment of acute leukemia.For those with relapse or refractory disease,more intensive therapeutic strategies should be given,such as allogeneic hematopoietic stem cell transplantation(allo-HCT).However,relapse continues to be the major reason for treatment failure.Residual leukemia cells in the bone marrow(BM)after treatment are the origins for relapse.Compared to relapse in extramedullary sites,it is extremely difficult to treat recurrence in the BM successfully due to insensitivity to current treatment regimens and poor prognosis.Thus,how to eradicate residual leukemia cells in the BM is critically important for improving treatment efficacy and long-term outcomes and for the final goal of curing acute leukemia.Chemokines and their receptors play very important roles in the development,migration,and maintenance of immune cells.Chemokine CXCL12 and its receptor CXCR4 not only participate in the development of hematopoietic cells,but also in homing to and retention in the BM microenvironment of hematopoietic stem cells(HSC).Furthermore,CXCL12/CXCR4 pathway is critical for the migration and residence of acute leukemia cells in the BM.BM establishes an immunosuppressive microenvironment via multiple mechanisms,such as immune cells,cytokines,in order to protect HSC from damage to maintain normal and emergency hematopoiesis.The same environment also protects leukemia cells from chemotherapeutic agents and molecular target drugs.Leukemia cells could be mobilized into circulation by CXCR4 blockade in both patients and mice with leukemia.Combination of CXCR4 blockade with chemotherapy or molecular target drugs could improve the elimination of leukemia cells in animal models and patients with acute leukemia.However,the efficacy is limited in clinical patients with leukemia,which may be associated with drug resistant leukemia cells or leukemia stem cells that are mobilized by CXCR4 blockade.Therefore,exploring strategies that can efficiently eradicate drug resistant leukemia cells or leukemia stem cells is very important for improving the efficacy of CXCR4 blockade.In our previous study,we found that CXCR4 antagonist could significantly enhance graft-versus-leukemia(GVL)effects of allogeneic lymphocyte infusion(ALI)in immunodeficient mice engrafted with human B-cell acute lymphoblastic leukemia(B-ALL)that is created by forcing expression of MLL-AF9 gene in human CD34+ HSC.However,there are some issues should be addressed.First,the leukemia cells are created in laboratory,which may raise the issue of cell specific responses and provide limited evidence.Additionally,immunodeficient mice are used which lack of intact immune system.Whether GVL effects could be enhanced in hosts with intact immune system is unclear.Moreover,more studies are still needed to determine if CXCR4 blockade will impact on the course of graft-versus-host disease(GVHD)or donor hematopoietic engraftment.The aim of this study is to determine whether CXCR4 blockade can enhance GVL effects of ALI with leukemia cells collected from clinical patients and of allo-HCT against murine leukemia cells in multiple animal models;to determine whether CXCR4 blockade will influence GVHD and donor hematopoietic engraftment after allo-HCT to provide direct evidence and reference for subsequent clinical trials.Methods:Models of patient-derived xenotransplants(PDX)were created with samples from patients with B-ALL.Leukemia cells became detectable in peripheral blood(PB)of mice examined by flow cytometry(FCM).ALI was performed on the following day after FCM analysis and CXCR4 antagonist was injected after the activation of allogeneic T cells.Leukemia cells in PB and/or tissues were detected by FCM after treatment.With multiple animal allo-HCT models and murine cells of T-cell ALL(TALL)and acute myeloid leukemia(AML)as target cells,the enhancement of GVL effects by CXCR4 blockade was tested.Animal models of acute and chronic GVHD were established,and the impact of CXCR4 blockade on GVHD was evaluated.Mouse allo-HCT model was used to assess the influence of CXCR4 blockade on donor hematopoietic engraftment in recipient mice.Results:1.The BM leukemia cells of patients with B-ALL express high levels of CXCR4,and CXCR4 antagonist AMD3100 could mobilize B-ALL cells out of BM into circulation in PDX models,and the highest levels of leukemia cells in PB occurred 3 hours after AMD3100 injection.2.With PDX models created using samples from three patients with B-ALL,one or two rounds of ALI plus AMD3100 was performed.The results showed that B-ALL cells in the BM were resistant to ALI,and AMD3100 could significantly enhance GVL effects of allogeneic T cells against B-ALL cells of patients,leading to efficiently eradicate leukemia cells,including those protected in by the BM microenvironment.3.When high levels of leukemia cells were detected in PB of PDX mice,ALI could be performed after the mice were pretreated with chemotherapy.CXCR4 blockade also could be observed to enhance GVL effects to eliminate leukemia cells,including these protected in by the BM microenvironment.4.Murine T-ALL and AML cells express high levels of CXCR4 on their surfaces.Mobilization of leukemia cells by AMD3100 into PB also could be observed and 3 hours after AMD3100 injection is time point of maximum mobilization responses.With mouse allo-HCT models,AMD3100 was proved to be very efficient in enhancing GVL effects.Especially,when the leukemia burden is low at transplantation,recipient mice treated with AMD3100 could achieve a very high rate of leukemia remission,and approximately 85% of mice were long-term survivors.5.With mouse models of acute and chronic GVHD,no significant difference was observed in terms of bodyweight change,survival,clinical GVHD scores,and pathological findings of GVHD target organs between mice treated with AMD3100 and PBS.6.With mouse allo-HCT models of non-myeloablative conditions,AMD3100 was proved to promote engraftment of donor hematopoietic stem/progenitor cells in the BM of recipient mice.Conclusion:Both patients-derived and murine leukemia cells expressed high levels of CXCR4 on their surfaces,and CXCR4 blockade could mobilize leukemia cells from BM into circulation.Enhancement of GVL effects by CXCR4 blockade could be observed after both ALI and allo-HCT,leading to achieving high rate of leukemia remission.Short term of CXCR4 blockade had no significant impact on GVHD after allo-HCT,but could promote the engraftment of donor hematopoietic stem/progenitor cells.