Effection Of TJU103 And CTLA4-Ig On Chronic Graft-Versus-Host Disease And GVL After Donor Lymphocyte Infusion

Background and ObjectivesAccording with the progress of technique of allogeneic hematopoietic stem cell transplantation(allo-HSCT),it is a potentially curative therapy for many haematological malignancies,severe aplastic anemia,inherited disorders of blood cells and solid tumor.But graft-versus-host disease(GVHD),especially chronic graft-versus-host disease(cGVHD),is an increasingly common complication of allogeneic stem cell transplantation(alloSCT).Its pathogenesy is not clear,severity and extent is not prognosticated,and there are not efficacious strategies to management and prophylaxis.So the research of Allo-HSCT has focused on seeking an effective way to induce immunotolerance,drease chronic GVHD and reserve graft-versus-leukemia(GVL) effection.To establish a standardized animal model is the base on it.The theory that immuno-activation of T cells requires two signals from antigen presenting cells(APC) and the molecular basis and the net of signal transmission has been elucidate,have provide new target to induce immunotolerance to the specific antigen in order to decrease cGVHD and preserve GVL.So the goal of this research is to(1) establish a murine model of cGVHD induced by DLI with MHC-haplo-identity,(2) explore the feasibility and the efficiency of TJU 103 or(with) CTLA4-Ig to chronic GVHD,(3) explore the feasibility and the efficiency of TJU103 or(with) CTLA4-Ig to GVL effect by inoculation FBL-3 cell line in 100 days after DLI,(4)detect the effects of proliferation and cytotoxicity of donor T cell sitimulated by splenocyte of hosts by MLC.The aim of this research is to explore the effect of TJU103 and CTLA4-Ig on chronic GVHD and GVL effect after donor lymphocyte infusion and give clues to prevent and treat cGVHD clinically.Methods1.To induce murine model of chronic GVHD after donor lymphocyte infusion with MHC-haplo-identity.The dornors are inbred strain male Balb/c^H-2d,C57BL/6J^H-2b mice at age of 8-10 weeks,the host are inbred strain female CB6F1(Balb/cxC57BL/6J F1^H-2d/b) mice at age of 6-8 weeks.Dornor single splenocyte suspensions were prepared from the spleens of Balb/c or C57BL/6J mice in PBS.cGVHD was induced by injection of 3×10⁷,6×10⁷,9×10⁷ Balb/c cells i.v.into CB6F1 mice.Control mice was injected with RPMI-1640 into age-matched mice and injected i.v.with 6×10⁷ syngeneic C57BL/6J splenocytes.Transplant recipients were killed for analysis of parental cell engraftment by PCR in 2,5,8,12week,monitored for weight loss,hair loss,dry skin in tail and ears,diarrhea,and huncture every 3 days.Additionally,cGvHD was scored through histologic analysis of tissue sections using previously reported criteria.The significance of differences between clinical observation index and pathological score was calculated by binary logistic regression to find out the distinctive clinical index of the model and make the model standardized. 2.The effect of TJU103 and/or CTLA4-Ig to the chronic GVHD in vivo.On the basis of foregoing marine model of chroic GVHD,A dose of 6×10⁷/0.5 ml/mouse splenocyte was used to induce chronic GVHD,and TJU103,CTLA4-Ig and TJU103+CTLA4-Ig were given to interfere it before and after infusion,Positive control mice received 6×10⁷ /0.5 ml/mouse donor splenocyte,and negative control group was induced by injection of RPMI-1640 into age-matched mice.The recipients were killed for analysis of chimerism by PCR in 2,5,8,12week,monitored for weight loss,hair loss and dry skin in tail and ears every 3 days.Additionally,chronic GvHD was scored through histologic analysis of tissue sections using previously reported criteria.The score of clinical manifestation and tissue histopathology were calculated to evaluate the influence of all interference to chronic GVHD.3.The effect of TJU103 and CTLA4-Ig to the GVL in vivo.Some CB6F1 mice of each group that had previously received transplants of 6×10⁷ /0.5 ml/mouse splenocyte were infected with 5×10⁵ /0.5 ml/mouse intraperitoneally on day 100 after transplantation randomly.The live time was recorded and tissue histopathology were checked to observe infiltration of leukemic cell when the mice were dead in order to find out the reason of death.4.The efficacy of proliferation and cytotoxicity to the haplotype ntismatched graft and a Friend's cell line was investigated by mixed lymphocyte culture.One CB6F1 mouse of each group randomly were killed for MLC at 35 and 85 day after influsion,Stimulator cells were splenocytes from CB6F1 mice of all group treated with MMC.Responding T cells from wild Balb/c mice were prepared from pooled spleens,filtered,and washed with PBS.Cultures were incubated at 37℃with 5%CO2 for 5 days.Pooled effector cells were harvested and counted on day 5 for use in the proliferation and cytotoxicity assays by MTT.Target cells were the splenocyte of each group untreated with MMC and FBL-3 cells,target and effector cells were incubated together at 37℃for 4 h at various E:T ratios for MTT to evaluate cytotoxicity effect to the haplotype mismatched graft and a Friend's cell line. Statistical analysesThe SPSS software was used to manage the experimental datas.Binary logistic regression was use to illuminate the relations between clinical observation index and pathological score to find out the distinctive clinical index of the model.The significance of differences in cGVHD incidence was described by Kaplan survival curve and calculated by log-rank Mantel-Cox.Kruskal--Wallis and Mann-Whitney nonparametric test was used to compare the severity of chronic GVHD.Significance of differences of living time of leukemia mice was calculated by analysis of variance and SNK and LSD.Two independent samples test was used to compare the reproducibility of chronic GVHD.Significance of differences of growth rate and cytotoxicity between 35th and 100th was calculated by analysis of variance of randomixed block design and SNK and LSD.Differences were considered significant when P values below.05 were obtained.Result:1.Standardized murine model of chronic graft-versus-host disease was established after dornor lymphocyte infusion.(1) chimera analysis:It was demonstrated that injection of 6×10⁷,9×10⁷ splenocytes of Balb/c into immunocompetent CB6F1 mice resulted in a stable levels of parental cell engraftment,But injection of 3×10⁷ splenocytes of Balb/c did not result in a stable parental cell engraftment.Injection of B6 splenocytes into CB6F1 resulted in an initial burst of parental cell engraftment,however,between the 12th week of GVHD,the percentage of parental cells unexpectedly began to decrease.This decline in parental B6 cells continued until the end of the experiment,at which time the disease mirrored chronic GVHD.In contrast to the acute GVHD of others that die within 4-8 wk,we have observed B6-into-CB6F1 GVHD mice surviving for 100 days.(2) It was decided by pathological secore of liver,intestinal and skin that the mice affected chronic GVHD or not,the significance of differences between clinical observation index of skin lesion,weight loss,diarrhea,posture and pathological score was calculated by binary logistic regression,the likehood ratio test of the model showed that regression equation have the statistical significance(P=O.041),the variance related to affection is skin lesion and weight loss(Ps =0.000,Pw=O.037), According to statistical significance by P＜O.05,there are 95.8%chance for right prediction.So it was skin lesion and weight loss that contributed to this chronic GVHD for clinical observation.(3) GVHD clinical scoring:Injection of Balb/c splenocytes into CBF1 mice with distinct quantity resulted in the development of chronic GVHD with distinct degree expected.Control mice that received RPMI-1640 without additional splenocytes did not develop GVHD.Injection of B6 splenocytes into CB6F1 initiated an initial acute GVHD.However,the acute disease resolved,and the CB6F1 mice went on to develop chronic GVHD.The Kaplan survival curve of morbidity has statistical significance by log-rank test(P=0.000).There is statistical significance between B,C experimental groups and A experimental group,control group(P=O.O00),but compared B with C experimental groups and A experimental group with control group,these differences were no significant(P＞0.032) according with corrected size of test P＜0.0071.The severity of chronic GVHD has statistical significance by Kruskal--Wallis nonparametric test(P=0.015) among A,B and C groups.There is statistical significance between B experimental groups and C experimental group (P=0.004),but compared B with A experimental group and C with A experimental group,these differences were no significant(P＞0.015) according with corrected size of test P≤0.0125.There is no statistical significance in severity among group A,B,C in skin lesion and weight loss by the Kruskal- Wallis nonparametric test (P≥0.199).So infusion of 6×10⁷,9×10⁷ splenocyte of Balb/c can induce chronic GVHD successfully,infusion of 6×10⁷ is the best according to the clinical evaluationcriterion.(4)Tissue histopathology:All the mice were sacrificed at 100 days after infusion,injection of Balb/c splenocytes into CB6F1 mice can be detected the lesion of chronic GVHD with distinct degree,the incidence of A,B and C group are 4/12, 11/12,11/12.Control mice received RPMI-1640 did not develop GVHD.Injection of B6 splenocytes into CB6F1 initiated lesion of acute GVHD(the sample come from the mice used for chimera analysis),However,the CB6F1 mice developed lesion of chronic GVHD at 100 day after infusion.The severity of chronic GVHD has statistical significance by Kruskal-- Wallis nonparametric test(P=0.024) among A,B and C groups.There is statistical significance between A experimental groups and B experimental group(P=O.OIO),but compared B with C experimental group and A with C experimental group,these differences were no significant(P≥0.023 ) according with corrected size of test P≤0.0125.There is no statistical significance in severity among group A,B,C in liver,skin and intestine by the Kruskal--Wallis nonparametric test(P≥0.070).So infusion of 6×10⁷,9×10⁷ splenocyte of Balb/c can induce chronic GVHD successfully,infusion of 6×10⁷ is the best according to the pathological evaluation criterion.2.The effect of TJU103 and CTLA4-Ig for the chronic GVHD in vivo.(1) chimera analysis.Balb/c splenocytes into CB6F 1 resulted in the engraftment pattern expected for chronic GVHD,with stable engraftment of parental lymphocytes in the Group of cGVHD,TJU103,CTLA4-Ig,and TJU103+CTLA4-Ig at 2,5,8,12 weeks. (2) The reproducibility of chronic GVHD.Group cGVHD was compared with the foregoing model group injected with 6×10⁷ Balb/c splenocyte.There was no statistical significance in Kaplan survival curve by log-rank test(P=0.155).The severity of chronic GVHD had no statistical significance between two groups in skin lesion,weight loss and clinical score by the 2 independent samples nonparametric test (P≥0.143 ).The severity between two groups had no statistical significance in liver, skin and small intestine too(P≥0.061 ).So the chronic GVHD induced by infusion of 6×10⁷ Balb/c splenocyte could be reproduced.(3) Clinical manifestation of all group:Injection of Balb/c splenocytes into CB6F1 mice with or without incubation of TJU-103 and/or CTLA4-Ig resulted in the development of chronic GVHD with distinct degree.Control mice that received RPM1-1640 without additional splenocytes did not develop GVHD.The Kaplan survival curve of morbidity has statistical significance by log-rank test(P=0.000). There was statistical significance between TJU-103,cGVHD groups and control group(P=0.000),but compared CTLA4-Ig,TJU 103+CTLA4-Ig group with control group,the statistic was high difference between TJU-103,CTLA4-Ig, TJU103+CTLA4-Ig group and GVHD group(P =0.000);The score of chronic GVHD,skin and weight loss had no statistical significance by Kruskal--Wallis nonparametric test(P≥0.218) among TJU- 103,CTLA4-Ig,TJU 103+CTLA4-Ig and GVHD groups.So TJU-103,CTLA4-Ig and TJU103+CTLA4-Ig could change the Kaplan survival curve of morbidity,CTLA4-Ig and TJU103+CTLA4-Ig discreased morbidity of chronic GVHD(the morbidity of chronic GVHD group was 12/12,the morbidity of CTLA4-Ig group was 4/12;the morbidity of TJU103+CTLA4-Ig was 2/12),and TJU103 could delay the affected time(the medium time of chronic GVHD group was 6.8weeks,but the medium time of TJU103 group was 11 weeks ),but TJU-103,CTLA4-Ig,TJU103+CTLA4-Ig could not affect the severity of chronic GVHD.(4) Tissue histopathology:Injection of Balb/c splenocytes into CB6F1 mice could be detected the lesion of chronic GVHD with distinct degree in TJU-103, CTLA4-Ig,TJU103+CTLA4-Ig and GVHD groups,the incidence of GVHD, TJU-103,CTLA4-Ig and TJU103+CTLA4-Ig group were 12/12,9/12,4/12 and 12/12. Control mice received RPMI-1640 did not develop GVHD.The severity of chronic GVHD had statistical significance by Kruskal--Wallis nonparametfic test(P=0.019) among TJU-103,CTLA4-Ig,TJU103+CTLA4-Ig and GVHD groups,but there was no statistical significance between every two groups(P≥0.02 7) according with corrected size of test P≤0.0071.There was no statistical significance in severity among group TJU-103,CTLA4-Ig,TJU103+CTLA4-Ig and GVHD in liver and intestine by the Kruskal--Wallis nonparametric test(P≥0.183),but it was high difference in skin(P =0.O10)and there was no statistical significance between every two groups(P≥0.012 ) according with corrected size of test P≤0.0071.So CTLA4-Ig and TJU 103+CTLA4-Ig discreased morbidity of chronic GVHD(the morbidity of chronic GVHD group was 12/12,the morbidity of CTLA4-Ig group was 4/12,the morbidity of TJU103+CTLA4-Ig was 2/12),but the effect on Severity of chronic GVHD of TJU-103,CTLA4-Ig,TJU103+CTLA4-Ig was not clear.3.The effect on TJU103 and CTLA4-Ig for the GVL in vivo.Some recipient mice were injectded with FBL-3 cell(a Friend's cell line) intraperitoneally at 100 days after transplantation in TJU-103,CTLA4-Ig, TJU103+CTLA4-Ig and GVHD groups,compared their live time.there is statistical significance by one-way ANOVA(F=43.897 P=0.000),it was no statistical difference between group TJU103 and control group(P=0.119),but it was high statistical significance between others(P≤0.042).There were leukemia cells infiltrated in liver,lung,spleen,kidney and myeloid.So chronic GVHD discreased the live time of recipients with FBL-3,there was no statical difference between TJU103 and control group,CTLA4-Ig and TJU103+CTLA4-Ig increased the live time of recipients with FBL-3,and TJU103+CTLA4-Ig was stronger.4.The efficacy of all groups on proliferative response of T cell,leathal effect on the haplotype mismatched graft and a Friend's cell line was investigated in mixed lymphocyte culture.There was statistical significance in growth rate of recipients'T cells sitimulated by donors'T cells by analysis of variance of randomixed block design by LSD in the 35^th and 85^th after transplation(F₃5 =286.355 P₃5 = 0.000,F₈5 =151.991 P₈5 = 0.000 ), the effect of group and time had interaction(F=17.432 P=0.000).Compared group TJU103 with group CTLA4-Ig,it was no statistical difference(P=0.064),but it was high statistical difference between others(P =0.000) in the 35^th after transplation, however,compared group TJU103 with group cGVHD,it was no statistical difference(P=0.614),but it was high statistical significance between others(P =0.000) in the85^th after transplation by LSD.The growth rate had no statistical significance between the 35^th and the 85^th in control groups,cGVHD group and CTLA4-Ig group(P≥0.155),but there was statistical significance in TJU103 and TJU103+ CTLA4-Ig group(P≤0.001) by independent-samples T test.It was high statistical difference in cytotoxity of donor T-cell against recipient lymphocytes by randomized block design analysis of variance and LSD in the 35^th and 85^th after transplation(F₃₅=107.260 P₃₅=0.000,F₈₅=85.821 P₈₅=0.000),there was no statistical significance between group TJU103 and group CTLA4-Ig(P=0.909), but it was statistical significance between others(P≤0.005) in the 35^th after transplation,however there was no statistical significance between group TJU 103 and group cGVHD(P =0.614),but it was high statistical difference between others(P =0.000) in the85^th after transplation by LSD.The result demonstrated TJU103 inhibited the growth of T cell as well as CTLA4-Ig initially(0.3610±0.0213 vs 0.3296±0.0335) and weaken as chronic GVHD in the last(0.4988±0.0202 vs 0.5076±0.0307),meanwhile cytotoxity of donor T-cell against recipient lymphocytes of TJU103 changed from strong as CTLA4-Ig to weeken as chronic GVHD.There was no statistical significance in cytotoxity of donor T-cell against mouse erythroleukemia by analysis of variance of randomixed block design by LSD in the 35^th and 85_th after transplation(F₃₅=0.254 P₃₅=0.904,F₈₅=0.026 P₈₅=0.999),it was high statistical difference among concentration(F₃₅=58.757 P₃₅=0.000,F₈₅=29.188 P₈₅=0.000),and the effect of group and time had interaction(F₃₅=0.393 P₈₅=0.969, F₈₅=0.097 P₃₅=1.000).So there was no statistical significance in cytotoxity of donor T-cell against mouse erythroleukemia.Conclusion:1.Infusion of 6×10⁷,9×10⁷ splenocyte of Balb/c can induce chronic GVHD successfully,the lesion of skin was the main clinical manifestation,it was skin lesion and weight loss that contributed to this chronic GVHD for clinical observation,and the lesion of liver,skin and intestine was contributed to pathological index.2.Chronic GVHD induced by infusion of 6×10⁷VBalb/c splenocyte could be reproduced.CTLA4-Ig and TJU103+CTLA4-Ig discreased morbidity of chronic GVHD and TJU103 could delay the affected time but TJU-103,CTLA4-Ig, TJU103+CTLA4-Ig could not affect the severity of chronic GVHD.3.Chronic GVHD discreased the live time of recipients with FBL-3,there was no statical difference between TJU103 and control group,CTLA4-Ig and TJU103+CTLA4-Ig increased the live time of recipients with FBL-3,and TJU103+CTLA4-Ig was stronger.4.TJU103,CTLA4-Ig and TJU103+CTLA4-Ig inhibited the growth of T cell and the effect of TJU103 changed from weaken stage initially to strong stage at last, meanwhile,TJU103,CTLA4-Ig and TJU103+CTLA4-Ig stimulated the cytotoxity of donor T-cell against recipient lymphocytes,and the effect of TJU103 changed from strong to weeken.TJU103,CTLA4-Ig and TJU103+CTLA4-Ig could not affect on cytotoxity of donor T-cell against mouse erythroleukemia.CTLA4-Ig and TJU103+CTLA4-Ig increased the live time of recipients with FBL-3,and TJU103+CTLA4-Ig was stronger.Innovation of the current study:we established a standardized mouse model induced by DLI,on the base of the model we demonstrated CTLA4-Ig and TJU103+CTLA4-Ig discreased morbidity of chronic GVHD and TJU103 could delay the affected time,CTLA4-Ig and TJU103+CTLA4-Ig increased the live time of recipients with FBL-3;we found Chronic GVHD discreased the live time of recipients with FBL-3 the first time;we demonstrated the delaying the affected time of TJU103 related to diversity of the cytokinetics.Significance of the research:Our study provided a new insight into the understanding the effect of T cell activation in chronic GVHD,moreover,provided theoretical and experimental basis on regimen of treatment and prophylaxis for chronic GVHD.