| Objective : The aim of this study is to investigate the effect of anti-CD3 in combination with busulfan and fludarabine preventing GVHD while preserving graft versus leukemia(graft versus leukemia(GVL) in mouse haploidentical hematopoietic stem cell transplantation model,as well as to explore the GVHD preventing mechanism of this conditioning regimen.Methods: CB6F1 mice(F1 of BALB/C and 57BL/6) were used as the recipients.C57BL/6 mice were used as donors. CB6F1 mice were assigned to three groups(n=6 for each group):Anti-CD3 group(anti-CD3 in combination with busfan and fludabine plus intravenous of splenocytes 100*10^6 and T cell deleted bone marrow cells 5*106), SPL group(busfan and ludabine plus intravenous of splenocytes 100*10^6 and T cell deleted bone marrow cells 5*106, BM group(busfan and fludabine plus intravenous of TCD bone marrow cells 5*106 only),Donor-derived lymphocytes and bone marrow cells were confirmed by flow cytometry. GVHD was monitored by the loss in total body weight ? clinical scores and survival. Histology of skin, intestine, liver and spleen were used to analysis the servity of GVHD. The yield of doner CD4+?CD8+ T cells in GVHD target organs were detected by flow cytometry. The target organ for CD4 and CD8 cells infiltration were detected by flow cytometry; IFN-? and TNF-a expression of CD4 and CD8 T cells in liver and spleen were also detected by flow cytometry.For GVL experiment, 50*106 splenocytes were used in transplantation, as well as0.5*106BCL1 cells were injected iv on the same day. After transplantation 3-4weeks. Flow cytometry was used to detect BCL1 cell of the recipient in peripheral blood, spleen and bone marrow.Results:(1) Establishment of Haplo-HSCT a GVHD mouse model: anti-CD3(5ug/g)13 days and 8 days before transplantation and busfan 15ug/g and fludabine 10ug/g before transplantation 5-2 days for twice intraperitoneal injection. Following the chemotherapy regimen and grouping method, lethal GVHD can be induced in the mice transplanted with 100*106 splenocytes and 5*106 T cell deleted bone marrow cells,showing lost of body weight, hunch back, diarrhea and hair lost.Engraftment was confirmed by flow cytometry, measuring by donor-derived CD4+lymphocytes, CD8+ lymphocytes.(2) Effect of anti-CD3: The mice of anti-CD3 group have a longer survival than those of SPL group. Furthermore, Anti-CD3 group markedly reduced lymphocyte infiltration and tissue damage in GVHD target organs.(3) anti-CD3 pretreatment for the prevention of GVHD mechanism:donor CD4 + and CD8 + T cells in GVHD target organ in Anti-CD3 group were significantly lower than those in SPL group; Seven days after transplantation,the expression of IFN-?and TNF-a of lymphocytes in spleen and liver of Anti-CD3 group was significantly reduced.(4)Systemic leukemia model: the CB6F1 mice were established by cyclophosphamide pretreatment, 3-4 weeks after intraperitoneal injection of BCL1 cells, and BCL1 cells were detected by flow cytometry.(5)Establishment of GVL model: spleen(50*106) and BCL1 cells(0.5*106); were injected on d0; 3-4weeks after transplantation, the BCL1 cells in spleen?peripheral blood and liver of Anti-CD3 group is not detectable.Thus,Anti-CD3 pretreatment still retain GVL effect.Conclusion:1. Preconditioning with anti-CD3 monoclonal can effectively prevent GVHD in mouse haplo-HSCT model.2. The preventing effect is associated with the inhibition of doner CD4+ ?CD8+ T cells infiltrating into the target tissues of GVHD.3. The prevention of GVHD is also associated with the reduction of IFN-?and TNF-a secretion.4. Anti-CD3 monoclonal can effectively prevent GVHD while preserve GVL in mouse haploidentical hematopoietic stem cell transplantation model... |
PDF Full Text Request