To Investigate The Effect Of High-dose PBSC As A Graft On AGVHD In Elderly Mice RIC-haplo-PSCT Model And Its Related Mechanism

Objective:To study the occurrence of acute graft-versus-host disease（aGVHD）in elderly patients with hematological malignancies treated with high-dose non-T cell-depleted（non-TCD）peripheral blood hematopoietic stem cells（PBSC）as graft and the related risk factors,and to explore the related mechanisms by which the high-dose non-TCD PBSC as grafts affects the aGVHD,in order to continually optimize the unique transplantation modality of our center and provide more possibilities for the treatment of elderly patients with hematological malignancies.Methods:Part I:We analyzed patients with hematological malignancies aged≥50 years treated with high-dose non-TCD PBSC as grafts for reduced-intensity conditioning haploidentical hematopoietic stem cell transplantation（RIC-haplo-PSCT）at our center from January 2014 to June 2022,who were infused with mononuclear cells（MNC）12-20×10⁸/kg and CD34+cells≥3×10⁶/kg,to analyze the occurrence of aGVHD and the risk factors affecting its occurrence,as well as engraftment,recurrence,and survival,and to provide evidence-based medical evidence for a unique transplantation model in our center.Part II:An aged mouse model of aGVHD after non-myeloablative haplo-PSCT was established.C57BL/6（H-2^b）male mice aged 6-8 weeks were used as donor mice and C57BL/6（H-2^b）×BABL/C（H-2^d）female CB6F1(H-2^b×d)mice aged 12-14 months were used as recipient mice.Different doses of radiation intensity were set.The radiation dose of non-myeloablative conditioning regimen was preliminarily determined.The recipients were randomly divided into control group,group A,group B and group C.Peripheral blood mononuclear cells（PBMC）and splenocytes were infused with 0.5×10⁷,1.0×10⁷and 2.0×10⁷ cells,respectively,through the tail vein within 4 hours after irradiation,and the control group was infused with the same amount of saline.After transplantation,the survival and clinical manifestations of aGVHD were observed in each group of mice,and the blood routine of mice was monitored to assess the damage of target organs such as skin,liver and intestines,and the post-transplantation aGVHD model was successfully constructed.Part III:On the basis of successful construction of aGVHD model after non-myeloablative haplo-PSCT in aged mice,the effect of infusion of different doses of PBMC on aGVHD and related mechanisms were investigated.C57BL/6（H-2^b）male mice at 6-8 weeks of age were used as donor mice,and CB6F1(H-2^b×d)female mice at 14 months-16 months of age were used as recipient mice,and regular-dose and high-dose groups were set up,in which the regular-dose group was treated with PBMC 1×10⁷,2.0×10⁷ of splenocytes,and basiliximab 1mg/kg,while the high-dose group was treated with PBMC 3×10⁷,4.0×10⁷ of splenocytes,and 2mg/kg of basiliximab.The survival and blood changes of mice in each group were observed after transplantation to assess the damage of the target organs of GVHD.Flow cytometry was also used to monitor the changes of CD4+T cells,CD8+T cells,myeloid-derived suppressor cells（MDSCs）,and the co-stimulatory molecules PD-1and CD28 after transplantation.Immunohistochemistry was used to observe the expression of PD-L1 and CD80 in the intestine.The expression of PD-1/PD-L1 and CD28/CD80 in intestinal tract was detected by Western blot.Results:Part I:A total of 40patients were included in this study,with a median age of 54.5 years and a median follow-up time of 29.5 months.The median doses of MNCs,CD34+cells and CD3+cells infused were 14.73×10⁸/kg,8.75×10⁶/kg and 6.05×10⁸/kg,respectively.The incidence of grade II-IV aGVHD was 21.5%（95%CI:5.9-34.5%）,and the incidence of grade III-IV aGVHD was 9.4%（95%CI:0-17.1%）.Cox regression analyses of the relevant risk factors affecting aGVHD showed no significant correlation.The 3-year cumulative incidence of c GVHD and extensive c GVHD was 28.0%（95%CI:9.7-42.6%）and 5.3%（95%CI:0-12.3%）,respectively.The 3-year disease-free survival（DFS）and overall survival（OS）were 72.5%（95%CI:59.1-88.9%）and 76.6%（95%CI:63.2-92.9%）,respectively.Part II:The body weight,activity and bone marrow suppression of mice in each group were decreased after different doses of radiation.The hematopoietic function of the control group recovered spontaneously during the observation period.The mice in group C showed a continuous decrease in blood routine after transplantation,which was lower than that of the other groups（P<0.05）.On+21d after transplantation,the cell chimerism rates of mice in groups A,B and C were all above 95%.Starting from+10d post-transplantation,mice in group C all showed typical aGVHD manifestations such as hair loss,bowed back,and diarrhea in varying degrees,and their aGVHD incidence rate was 100%.Pathologic examination on+21 days after transplantation showed that aGVHD pathological changes were seen in the skin,liver,and intestines of mice in group C,and their pathohistological scores were higher than those of the other groups（P<0.05）.Part III:There was no statistically difference in the clinical scores of aGVHD between the two groups of mice（P>0.05）.The blood routine of mice in the high-dose group began to recover slowly at+7d post-transplantation,while that of the standard-dose group began to recover at+14d post-transplantation.The donor cell chimerism rate of mice in the high dose group was higher than that in the conventional dose group at+7d post-transplantation（P>0.05）.CD4+T cells and CD8+T lymphocytes were detected in peripheral blood at different time periods after transplantation,and the differences between the two groups were not statistically（P>0.05）.The number of MDSCs in the spleen was low in the early post-transplantation period,and then continued to increase,until+21d the MDSCs in the high-dose group were significantly higher than those in the standard-dose group,and the subpopulations of MDSCs were detected.The most significant change in the impact on the number of MDSCs was the G-MDSCs.At+21d post-transplantation,the expression of PD-1 on the surface of the CD4+T cells in the high-dose group was higher than that in the standard-dose group（P<0.01）.The expression of PD-1/PD-L1 in the intestinal tissues of mice in the high-dose group was significantly higher than that in the standard-dose group,and the expression of CD28/CD80 was significantly lower than that in the standard-dose group.Conclusions:（1）High-dose non-TCD PBSC as grafts for the treatment of elderly patients with hematological malignancies has achieved encouraging results,with a lower incidence of post-transplant aGVHD,high engraftment and better survival.（2）For aged CB6F1 mice,a non-myeloablative pretreatment dose of 6 Gy TBI with linear accelerator X-ray was administered.Aged CB6F1 mice infused with PBMC（1×10⁷/each）and splenocytes（2.0×10⁷/each）were able to successfully induce an aGVHD model,providing a reliable preclinical model for use as a study of the mechanisms associated with aGVHD development.（3）Infusion of high-dose non-TCD PBSC combined with an intensive GVHD prophylaxis regimen significantly attenuated the occurrence of aGVHD,which may be related to affecting the level of MDSCs,may also modulate the expression of negative co-stimulatory molecules PD-1/PD-L1,positive co-stimulatory molecules CD28/CD80,thereby reducing the severity of aGVHD.