Studies On The Effects Of Adoptive Immunotherapy Post Mixed Hematopoietic Stem Cell Transplantation By Genetic Mark Technology

Owing to graft-verus-leukemia(GVL) effects,allogeneic hematopoietic stem cell transplantation(allo-HSCT) has an advantage of low relapse rate,but a high incidence of transplant-related mortality.Due to the complications such as graft-versus-host disease(GVHD),infection and so on;On the other hand,most patients have no chance to receive allo-HSCT because of HLA restriction.Up to now,autogeneic hematopoietic stem cell has been applied nearly for forty years in clinics,it has the advantage of unlimited donor,fewer complications,but it has a high relapse risk for lack of GVL effects.Relapse is the major reason for failure of auto-HSCT, decreasing relapse is the key point to increase therapeutic effect.Our previous research showed:autologous marrow mixed with HLA-haploidentical allogeneic stem cell transplantation(Mixed-HSCT) could exploit the advantage of both sides in a certain degree,overcome their shortcomings,develop mixed chimerism,reduce GVHD,and keep GVL effects.By leukemia rat model using K562 cell line with gene marker and BALB/C mouse,we studied the therapeutic effects and the forming of chimera of Mixed-HSCT and post transplant donor lymph cell infusion combined IL-2 for acute myeloid leukemia,provided evidence for clinical application;Furthermore,we also observed clinical therapeutic efficacy of mixed-HSCT.1.Methods:(1) Gene transfer:Cultured PA317-GCGPXSN,NIH3T3 and K562 cell lines;prepare virus supernatant with PA317-GCGPXSN cell line;Performed the gene transfer after stimulating K562 cells with rhIL-3,rhIL-6 and rhSCF;Evaluate efficiency of gene transfer by flow cytometry and assess the NeoR gene by PCR.The K562 cells having fulfilled gene transfer were defined as K562+ cells. (2) BALB/C mouse model:SPF(specific-pathogen free) BALB/C mice were irradiated 2Gy or 3Gy for 24 hours respectively according to experiment groups,then injected into log phase growth K562+ cells by vena caudalis.observed general state of health,survive time,bone marrow cell classification,and peripheral blood leucocyte count,classifcation, subset,related organ pathology checking;detected K562+ cells by flow cytometry;detected tissue infiltration by PCR.(3) Cultured bone marrow cells of BALB/C×C57BL first filial generation(F1) mice according to routine methods,detected T cell subsets,NK cells,CD34+ cells.After gene transfer,detected the efficiency of gene transfer by flow cytometry,detected NeoR gene by PCR.The F1 ceils having fulfilled gene transfer were defined as F1+ cells.(4) Animal experiment:Seven days later,the leukemia mice prepared as above methods were performed ABMT or MBMT(allogeneic bone marrow cells were reinfused 1h after ABMT),after irradiated 6Gy for 24 hours.The mice of MBMT group were reinfused F1 cells every 7 days,and administered IL-2 by intraperitoneal injection every other day.Killed the surviving mice after 4 week observation,observed peripheral blood and bone marrow cell morphous,detected cell subsets,GFP+ cells,NeoR gene,examined the liver,spleed pathology and detected GFP+ cells and NeoR gene of their tissue homogenate.(5) 13 patients with acute myeloid leukemia(AML),in complete remission(CR),were undergone Mixed-HSCT,after hemopoiesis recovery,administered donor lymphocyte infusion(DLI) for 2～7 times.Another 11 AML patients also in CR period were undergone Mixed-HSCT,administered no special treatment post transplantation.2.Results:(1) The gene transfer efficiency in K562 cells:fluorescence intensity value measured by flow cytometry was 0.56%in K562- cells,and 77.16%in K562+ cells,there is significant difference between them(P＜0.001>.The special segment of NeoR gene was amplificated by PCR,which showed that retroviral vector plasmid contained GFP and NeoR gene had been transferred and integrated into the genome of K562 cells.(2) The preparation of leukemia model①The effects of irradiation on immune function of mice:after the mice of E group were irradiated 2Gy,their CD3+,CD4+,CD8+,CD16+CD56+ cells in peripheral blood decreased progressively,up to third week,begun to recover gradually,four weeks later recovered to normal.In F group,after irradiated 3Gy,the CD3+,CD4+,CD8+,CD16+CD56+ cells also decreased progressively,while up to fourth week,begun to recover gradually,five weeks later recovered to normal.At the same time point,the extent that cell subsets in mice irradiated by 3 GY decreased was more notable than that by 2Gy(P＜0.05).After irradiation, the spleed,thymus diminished compared with normal mice,their weight lightened and four weeks later recovered to the level before irradiation,the weight of liver had no change.②The incidence:the BALB/C mice(E,F group) irradiated by 2 or 3Gy but without K562 cells infused had no natural death after observed for four weeks.The BALB/C mice(A,B,C,D group) irradiated by 2 or 3Gy and infused 2×10~6,5×10~6 K562 cells fell ill 5～7 days later,all of mice in A group died within 30 days,C group within 24 days,B group within 23 days,D group within 17 days;there is significant difference in survival time,which is shorter than that of normal control group(P＜0.01).The weight in four groups decreased significantly than that in normal control group(P＜0.01).The incidence of leukemia in processed mice was 100%,and had no natural relief.With the increase of infused leukemia cell population and the increase of irradiation dose,survive time of mice shorten gradually,Furthermore the percentage of myeloblast or premyelocyte in peripheral blood and bone marrow increased gradually.Tumour cell infiltration was confirmed by pathology-scopy.The results detected by flow cytometry coincided with that,the existence of NeoR gene was verified by PCR.(3) The gene transfer efficiency in F1 cells:Fluorescence intensity value measured by flow cytometry was 0.63%in F1(-) cells,and 76.04%in F1(+) cells,there is significant difference between them(P＜0.001>.The special segments of NeoR gene were amplificated by PCR,which showed that retroviral vector plasmid contained GFP and NeoR gene had been transferred and integrated into the genome of F1(+)cells.(4) Bone marrow transplant on mice①The cell population of bone marrow transplant:In the experiment group,of the transplanted autologous bone marrow cells,the percentage of CD34+ cells is 1.32%;CD3+ cells,26.56%;CD4+ cells,13.44%;CD8+ cells,3.09%;CD16+CD56+ cells,7.53%.Of the transplanted allogeneic bone marrow cell(F1+),the percentage of CD34+ cells is 1.51%;CD3+ cells,61.41%;CD4+ cells,39.57%;CD8+ cells,27.61%;CD16+CD56+ cells,9.59%;Of the transplanted allogeneic bone marrow cell(F1-),the percentage of CD34+ cells is 1.49%;CD3+ cells,65.86%;CD4+ cells,40.13%;CD8+ cells,19.16%;CD16+CD56+ cells,9.70%.There is no significant difference between F1(+)and F1(-)cell subsets(P＞0.05), that means:there is no difference about all kinds of cell population transplanted to each experiment mouse.②Therapeutic effects:All of mice in leukemia model group(A group) died within 20 days;the mice of leukemia model irradiated 6 GY(F group),died within 14 days.There was 11%～64%myeloblast or premyelocyte in peripheral blood or bone marrow of dying mice in A group;while in F group,peripheral blood or bone marrow cells were rare,mainly lymph cells.GFP and NeoR gene were detected in bone marrow and peripheral blood of both A group and F group dying mice.The T cell subsets and NK cells were scarce in dying mice of both groups.There were 3～6 mice surviving above 28 days in other experiment groups,the quantity and morphous of survival mouse's bone marrow and peripheral blood cells identified with normal mice,T cell subsets and NK cells of peripheral blood had recovered to normal range,there was no significant difference among each group(＞P0.05).GFP and NeoR gene were detected in survival mice infused F1(+) cells.Tumour cell infiltration was not found in liver or spleed of survival mice.GFP and NeoR gene fragments were not detected in tissue homogenate,which demonstrated K562+ tumour cells disappeared in survival mouse liver and spleed.(5) Clinical therapeutic effects:In observation group,all of patients undergone Mixed HSCT had hematopoietic reconstitution,no GVHD occurred.three M2a patients,who received once,twice,twice DLI+IL-2 therapy,relapsed and died at 3,4,7 months post therapy respectively.One M2a patients receiving twice DLI+IL-2 therapy suffered myelodysplastic syndrome 1 year after therapy,and died of cerebral hemorrhage at last.One M5 patient emerged unknown hyperthermy and twitch after receiveing once DLI+IL-2 therapy,and died of statural epilepticus.Other 8 patients had been disease-free survival(DFS) with follow-up 1～5 years.Among them,one M4 patient,one M5 patient were found to have mixed chimera.The long term survival was 61.5%(8/13).In control group,2 cases of M2a,2 cases of M4,1 case of M5 relapsed and died 1～7months post transplantation,one M3 patients died of cerebral hemorrhage at 25 days post transplantation.Other 2 cases of M3,2 cases of M4,1 case of M5 patients had been disease-free survival(DFS) with follow-up 3～5 years.Among them,the later three cases had mixed chimera formed.The long term survival was 45.4%(5/11).3.Conclusion:(1) The retrovirus contained of GFP gene and NeoR gene can be successfully transferred into K562 cell line and bone marrow cells of BALB/C×C57BL F1 mice.(2) The leukemia mouse model can be made by infusing K562 cells by vena caudalis into BALB/C mouse after irradiated.(3) Long term survival can be promoted when the leukemia mice are administered DLI combined with IL-2 therapy post Mixed-HSCT.(4) It shows according to clinical application:long term survival can be promoted when the AML patients are administered DLI combined with IL-2 adoptive immunotherapy post Mixed-HSCT.