| [Objective]Patients with relapsed/refractory diffuse large B-cell lymphoma（DLBCL）have a very poor prognosis.High-dose chemotherapy combined with autologous hematopoietic stem cell transplantation（ASCT）is a classic salvage regimen for the treatment of relapsed/refractory DLBCL,but only 26-64%of patients can achieve remission,and nearly half of the patients will experience relapse after remission.In recent years,CART therapy has achieved remarkable success in the treatment of relapsed/refractory DLBCL,but 40-50%of patients who obtained remission after CART treatment will still experience relapse or progression.In order to improve the efficacy of relapsed/refractory DLBCL,this study investigated the efficacy of ASCT bridging to CART（ASCT-CART）in the treatment of relapsed/refractory DLBCL patients,and compared it with ASCT alone.Based on the significant efficacy of ASCT-CART in the treatment of DLBCL,we attempt to transfer the CAR gene into hematopoietic stem cells（HSC）to form CAR-expressing HSC（CAR-HSC）,and to explore the hematopoietic reconstitution and differentiation of CAR-HSC in animals and its efficacy and safety for treating DLBCL.[Methods]1.The retrospective study included 70 patients with relapsed/refractory DLBCL who received autologous hematopoietic stem cell transplantation（ASCT）alone or ASCT bridging to CART（ASCT-CART）in the Department of Hematology of the First Affiliated Hospital of Naval Military Medical University from 2016 to 2021.Twenty-four were treated with ASCT-CART and 46 were treated with ASCT.Patients included were who did not achieve CR after 6 cycles of standard R-CHOP chemotherapy or 4 cycles of standard R-CHOP chemotherapy and 2 cycles of second-line chemotherapy,and patients who relapsed or progressed within 12 months after achieving CR or PR.The dichotomous data involved in the study were analyzed by the chi-square test,the measurement data by the t test,and the survival data by the Kaplan-Meier survival analysis and log-rank test.P<0.05 was considered statistically significant.2.Basic research（1）Stem cell sorting and CAR-lentivirus vectors transfection.Mouse bone marrow hematopoietic stem cells were sorted by magnetic beads,and the hematopoietic stem cells were transfected with CAR-lentivirus vectors to form CAR-HSC,and CAR-HSC transfection efficiency was detected by flow cytometry.（2）Stem cell differentiation and hematopoietic reconstitution.The colony formation ability of CAR-HSC was explored through the CAR-HSC in vitro colony formation assay;The hematopoietic reconstitution and differentiation after CAR-HSC ASCT were used to monitor the proportion of peripheral blood cells and the proportion of CAR-positive immune cells.HSC differentiation and hematopoietic reconstitution in mice.（3）Establishment of ASCT model.C57/BL6N mice with CD45.1 phenotype were used as donors,and C57/BL6N mice with CD45.2 phenotype were used as recipients.The myeloablative precondition of the recipients was lethally irradiated with Co60,followed by the donor hematopoietic stem cells infusion.The success of the mouse ASCT model was assessed by monitoring the mouse hematopoietic reconstitution and chimerism rate（proportion of CD45.1 positive cells）.（4）Establishment of DLBCL model.The mouse melanoma cell line B16 with low immunogenicity was used to overexpress human CD19 molecule.B16-CD19 cells were constructed,and subcutaneously implanted into tumors to construct a mouse tumor model similar to human DLBCL.（5）CAR-HSC autologous transplantation for DLBCL model.After the recipient lethally irradiated with Co60,B16-CD19 tumor cells were subcutaneously implanted,and then CAR-HSC was reinfused to evaluate CAR-HSC efficacy by observing tumor growth rate and mouse survival,to assess the toxic and side effects of CAR-HSC like cytokine release syndrome by detecting peripheral blood cytokines,and to identify CAR-positive immune cells generated by CAR-HSC differentiation cellular remodeling of the tumor microenvironment by detecting immune cells infiltrated in tumors by flow cytometry and immunohistochemistry,.[Results]1.In clinical studies,in patients with relapsed/refractory DLBCL in ASCT-CART group and ASCT group,the complete remission rates were 75%and 33%,respectively（P=0.001）,and the partial remission rates were 167%and 57%（P=0.001）.The 3-year PFS was 81%and44%in the ASCT-CART and ASCT groups（P=0.032）.The 3-year OS was 80.1%and 69%in the ASCT-CART and ASCT groups,respectively（P=0.541）,and there was no significant difference in OS between the two groups.The 3-year cumulative recurrence rate of ASCT-CART group and ASCT group were 15%and 56%（P=0.013）.2.4 weeks after autologous transplantation,all of bone marrow cell control group（BMC）,GFP-HSC positive control group and CAR-HSC group experienced hematopoietic reconstitution.Chimerism rates of BMC group,GFP-HSC group and CAR-HSC group were89.80±0.31%,91.65±1.62%and 88.80±3.02%,respectively（P=0.606）.3.After ASCT,the proportions of B cells in BMC,GFP-HSC and CAR-HSC groups were 49.28%±1.78%,49.63±3.69%and 31.80%±1.58%,respectively.The proportion of B cells in the CAR-HSC group was significantly lower than that in the BMC group（P=0.001）and the GFP-HSC group（P=0.001）.The proportion of neutrophils in BMC group,GFP-HSC group and CAR-HSC group were 13.18%±1.42%、19.78%±2.31%and 36.73%±4.45%,respectively.The proportion of neutrophils in the CAR-HSC group was significantly higher than those in the BMC group（P=0.0001）and the GFP-HSC group（P=0.02）.Other immune cells,including T cells,NK cells,and monocytes,were not significantly different among the three groups.4.CAR-HSC transplantation differentiate to peripheral blood cell contains9.34%±2.02%of CAR-positive cells,including 5.17±2.10%of CAR-T cells,13.79±4.27%of CAR-NK cells,6.27±1.98%of CAR-B cells,46.63±14.27%of CAR-granulocyte and15.48±2.82%of CAR-macrophage.5.CAR-HSC treated the DLBCL model,and the untransfected HSC were used as the control group（CTRL）.The types and proportions of immune cells in the tumor were detected by flow cytometry and immunohistochemistry 21 days after transplantation.The proportion of immune cells infiltrating the tumor in the CTRL and CAR-HSC groups were 24.20%±4.79%and 27.3%±3.70%,respectively（P=0.610）.The CAR gene m RNA level in the tumor of the CAR-HSC group was significantly higher than that of the control group.6.The proportion of CD3-positive T cells detected by flow cytometry in CTRL group and CAR-HSC group was 25.58±4.45%and 16.17%±3.68%,respectively（P=0.312）.RT-PCR detection of CD4 and CD8 m RNA levels in tumor tissue showed that CD8/CD4 m RNA level in the CAR-HSC group was significantly higher than that in the CTRL group（P=0.036）.7.The proportion and subtypes of tumor-associated macrophages（TAM）were detected by flow cytometry.The results showed that the proportion of TAM in the CTRL group and the CAR-HSC group were 5.53%±0.85%and 5.89%±2.60%,respectively.There was no significant difference between the two groups.difference.The M2/M1 ratio was1.68%±0.23%in the CTRL group and 0.94%±0.16%in the CAR-HSC group（P=0.009）.8.The tumor-associated neutrophils（TAN）in CTRL and CAR-HSC group were10.49%±1.80%（n=3）and 32.57%±5.71%,respectively（P=0.005）.9.Immunohistochemical detection of immune cells in the CTRL group and CAR-HSC group showed that the proportion of CD8-positive T cells in the CAR-HSC group was significantly higher than that in the control group（P=0.027）.Among tumor-infiltrating TAMs,the proportion of M2-type TAMs in the CAR-HSC group was significantly lower than that in the CTRL group（P=0.027）and the proportion of M1-type TAMs was significantly higher than that in the control group（P=0.001）.The proportion of infiltrated neutrophils in the CAR-HSC group was significantly higher than that in the control group（P=0.040）.10.The tumor volume of CAR-HSC group showed slower increasing than CTRL group（P=0.048）.The median survival time of mice in CTRL group and CAR-HSC group was 22（95%CI17-26）days and 25（95%CI24-25）days,respectively.The overall survival time of mice in the CAR-HSC group was significantly longer than that in the CTRL group（P=0.029）.[Conclusion]Compared with ASCT,ASCT-CART in the treatment of relapsed/refractory DLBCL significantly improves the CR rate,prolonged PFS,and significantly decreased the accumulative recurrence rate.The efficacy of ASCT-CART in the treatment of relapsed/refractory DLBCL is significantly better than that of traditional ASCT alone.In basic research,HSC transfected with lentivirus did not affect the differentiation and hematopoietic reconstruction functions of HSC in vivo.CAR-HSC remodels the tumor microenvironment,kill tumor cells,prolong the survival time of mice and has no serious treatment-related complications. |
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