| Acute myeloid leukemia(AML)is an aggressive hematologic malignancy that primarily affects the elderly population,with more than half of AML patients over 60years of age.Most elderly people cannot tolerate standard intensive chemotherapy due to their poor organ reserve function,underlying comorbidities and high rates of adverse cytogenetics,which makes the treatment of AML in the elderly a constant challenge.Moreover,the progression of AML alters the bone marrow microenvironment and suppresses normal hematopoiesis,ultimately leading to immune deficiency in patients.Therefore,the study of cellular immune function changes in elderly AML patients may provide new ideas for treatment and prognosis.Objective In this paper,through the comparative analysis of lymphocyte subsets of normal elderly AML patients after micro-transplantation(MST),explored the influence of age,risk of prognosis,relapse and clinical significance of MST treatment,and further analyzed the difference of TCR diversity under different treatment methods in elderly AML patients.On this basis,the study hoped to increase the efficacy of MST by using the precursor T cells induced in vitro.Methods Firstly,we reviewed and analyzed the cellular immune function of 25 healthy elderly patients who were examined in the Fifth Medical Center of the PLA General Hospital.Divided them into 10 cases aged 50-59,10 cases aged 60-69,and 5 cases aged70-79 by age,and statistically analyzed the differences and similarities of lymphocyte subsets in each group.Secondly,a retrospective analysis was conducted on 41 elderly AML patients who received MST treatment at the Fifth Medical Center of the People’s Liberation Army General Hospital from August 2014 to September 2018.Divided the patients into three groups by age:11 cases aged 50-59,18 cases aged 60-69,and 12cases aged 70-79.They were divided into 2 groups based on prognostic risk,with 24cases in the medium-low-risk group and 17 cases in the high-risk group.According to the pre transplant status,there were 3 groups:22 cases with initial treatment,15 cases with complete remission,and 4 cases with recurrence or no remission.We compared the immune function differences of newly treated elderly AML patients before and after MST treatment.We also analyzed the changes in lymphocyte subpopulations of patients in different age groups at different time periods within one year after reaching CR.Then,we analyzed the differences in immune function between the high-risk and medium to low risk groups of elderly AML after receiving MST treatment for CR,as well as the changes in immune function of these patients before and after MST treatment and during the relapse stage.We further used high-throughput sequencing technology to compare the TCR diversity of elderly AML patients under different treatments.Based on the above data results,we had designed animal experiments to increase the immune recovery effect of MST.Targeted differentiation of mouse bone marrow stem progenitor cells into precursor T cells was induced by Notch-DLL4 protein stimulation in vitro,and cell expansion was observed and identified by flow cytometry.In addition,this study preliminarily established an animal model of pre T cell optimized micro transplantation for immune recovery.In this experiment,CB6F1 mice were used as recipients without any pretreatment,and four groups were established.The first group received infusion of MNC and pre T cells,the second group received single infusion of MNC,the third group received single infusion of pre T cells,and the fourth group was a blank control group.After micro transplantation,peripheral blood from mice was collected at different time points for flow cytometry immune function testing.Results There was no statistically significant difference in the proportion of lymphocyte subsets between normal elderly patients aged 50-59 and those aged 60-69.Compared with those aged 70-79,those aged 50-59 have a higher proportion of CD4~+T cells(P=0.038)and a lower proportion of CD8~+T cells(P=0.011);The proportion of CD8~+T cells in individuals aged 60 to 69 is relatively low(P=0.007).After the first course of MST treatment,35 AML patients received CR and 6 had NR.From the beginning of MST treatment to the end of follow-up,there were 13 cases of sustained CR and 20cases of recurrence during this period.After MST treatment of CR,compared with the initial diagnosis,the CD3~+T cells of CR patients were significantly increased(P<0.05).After micro transplantation treatment,the CD3~+,CD8~+T cells,and activated T cells of elderly AML patients in three age groups were significantly higher than normal values within one year(P<0.05),while B lymphocytes and NK cells were consistently lower than normal values within one year after MST treatment(P<0.05).Within one year of micro transplantation treatment,CD3~+,CD8~+T cells,and activated T cells were significantly higher than normal values in both high-risk and low-risk groups(P<0.05).The low-risk group tended to stabilize earlier,while B lymphocytes,NK cells,and CD4/CD8 ratio were significantly lower than normal values(P<0.05).The overall recovery of CD4~+T cells and na?ve T cells in the low-risk group was better than that in the high-risk group.Compared with the continuous CR state after MST treatment,CD3~+,CD4~+T cells,CD4/CD8 were significantly decreased(P<0.05)during the recurrence of patients,and the proportion of CD8~+T cells,B lymphocytes,NK cells,and na?ve CD8~+cells increased.Compared with the initial diagnosis,NK cells,regulatory T cells,and activated T cells increase during recurrence.The diversity of TCR after micro transplantation treatment is the best,followed by chemotherapy,and the worst at the initial diagnosis of elderly AML.The number of pre T cells cultured in vitro using the DL4-Fc system increased by approximately 80 times on the 12th day.Most of the cells were identified by flow cytometry as pre T cells in the DN2-DN3 stage.Conclusion In this study,we found that there were differences in lymphocyte subsets among normal elderly people of different ages.The cellular immune function of normal people aged 50-59 and 60-69 was basically similar.The immune function of normal elderly people over 70 years old decreased significantly,which was mainly manifested by the decrease of CD4~+cells and the increase of CD8~+killer cells.In addition,this study also found that the lymphocyte subsets of elderly AML patients changed within 1year after micro-transplantation in CR state.MST promoted the recovery of CD3~+and CD8~+T cells and other killer cells.The activation and supplementation of CD4~+T cells over 60 years old were more obvious.Compared with the high-risk group,the overall recovery of immune cells in the low-risk group was better,including the recovery of CD4~+T cells and na?ve T cells.In conclusion,the immune function of patients after MST treatment was restored,and more killer cells could be activated,which indirectly reflects the role of MST cell therapy in mobilizing the immune function of recipients.The TCR of newly diagnosed EAML patients lacked diversity and was dominated by monoclonal proliferation.Compared with chemotherapy alone,micro-transplantation may improve the diversity of TCR repertoire more quickly and effectively.Finally,we successfully induced the differentiation and expansion of bone marrow-derived mouse stem progenitor cells into precursor T cells using the DL4-Fc system. |
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