Study Of T-cell Subsets In Peripheral Blood Of B-cell Acute Lymphoblastic Leukemia Patients And Their Effect On CD19 CAR-T Cell Therapy

Changes and Significance of T Cell Subsets in Patients with Acute B cell Lymphoblastic LeukemiaBackground Acute lymphoblastic leukemia(ALL)is one of the most common types of leukemia in children and adults.According to cell phenotype,ALL can be divided into acute B cell lymphocytic leukemia(B-ALL)and acute T cell lymphoblastic leukemia(T-ALL),and B-ALL is more common.The pathogenesis of B-ALL is complex.Gene mutation,genetic material abnormality and immune microenvironment are important factors.However,the immune microenvironment is always dynamic during the development of acute leukemia.The change of immune microenvironment can lead to change of immune cell subsets in the peripheral circulation.It means that immune cell subsets may indirectly reflect immune microenvironment.Therefore,it is necessary to further explore the expression of peripheral T cell subsets in B-ALL patients and their relationship with different disease periods and therapeutic effects,and finally provide a theoretical basis for optimizing clinical treatment and judging prognosis.Objectives To explore the distribution of peripheral blood T cell subsets in B-ALL patients and assessed their relationship with different disease periods and therapeutic effects.Methods The clinical data of 60 adult B-ALL patients were collected and analyzed.Among them,16 were newly diagnosed,20 were at remission and 24 were relapsed after remission.Using direct fluorescein to label cell surface antigens,peripheral blood CD4+T cells,CD8+T cells,CD4/CD8 ratios and Treg cell levels were measured by flow cytometry.Besides,40 healthy adults were selected as healthy controls.Results There were no significant correlation between T cell subsets and clinical characteristics such as age,sex,white blood cell count,therapeutic effect of induction therapy.CD3+ T cell,CD4+ T cell and CD4/CD8 ratio in peripheral blood were lower in patients with high-risk molecular or genetic abnormalities.However,CD4+ Treg cells were higher in patients with high-risk molecular or genetic abnormalities.Compared with the healthy controls,CD4+ T cell decreased in newly diagnosed(ND)group,increased when the disease reached remission,and decreased again when relapsed after remission.CD8+ T cell expression in the normal control group was the lowest among the four groups.Besides,CD4/CD8 ratio was significantly decreased in ND group.When these patients achieved remission,CD4/CD8 ratio elevated.However,when these patients relapsed again,CD4/CD8 ratio decreased once more.Compared with the normal control group and CR group,the proportations of Treg cell in the ND group and relapsed group were significantly increased.Besides,the expression of Treg cell in relapsed group was higher than that in the ND group.In addition,we also analyzed the T cell subsets of eight B-ALL patients at initial diagnosis and at complete remission.Our results showed that abnormal elevated Treg cells at initial diagnosis decreased significantly when the patients reached complete remission(P=0.001).However,there were no significant differences between CD4+T cells,CD8+T cells and CD4/CD8 ratio between the two groups.Conclusion In newly diagnosed B-ALL patients,CD8+T cells and Treg cells increased obviously,while the CD4/CD8 ratio reduced,suggesting that T cell immune dysfunction may be involved in the pathogenesis of B-ALL.When the patients achieved remission by chemotherapy,the abnormal T cell immune function could be partially recovered,indicating that effective treatment may improve the T cell immune imbalance in B-ALL patients.In addition,T cell immune imbalance reappeared when the disease recurred,indicating that the abnormality of T cell immune function may also be involved in the recurrence and progression of B-ALL.Influence of T cell Subsets on the effect of CD19 CAR-T therapy for relapsed/refractory B-ALLBackground Relapse/refractory B-ALL usually has poor prognosis and the therapy is limited.CD19 Chimeric antigen receptor T cells(CAR-T)therapy had high remission rate for relapse/refractory B-ALL.However,long-term follow-up revealed that 30%~50% of the patients had recurrence after remission.In addition,about 10%~20% of patients failed to this therapy.Recurrence and failure of treatment have become major challenges in the clinical application of CD19 CAR-T therapy.The effect of CAR-T therapy is related to the persistence of CAR-T cells in patients,and many factors such as immune status,tumor biological characteristics can affect the retention time of CAR-T cells.Our previous study has found that T cell immune function was related to the occurrence and progression of B-ALL,the influence of peripheral T cell subsets on CAR-T cell therapy in recurrent/refractory B-ALL patients needs to be further explored.Objectives To explore T cell immune function in peripheral blood of relapsed/refractory B-ALL patients before and after CAR-T cell infusion,and analyze its effect on the therapeutic efficacy of CAR-T cells.Methods Cell surface antigen was labeled by direct fluorescein method,and the levels of peripheral blood CD4+T cells,CD8+T cells,CD4/CD8 ratios and CD4+Treg cells in 46 recurrent/refractory B-ALL patients before CAR-T cells infusion and at 1 week after infusion were detected by flow cytometry.The differences of immune status before and after CAR-T cells infusion,and their relationship with clinical characteristics,adverse reactions,short-term efficacy,recurrence and long-term survival of CAR-T cell therapy were all analyzed.Results Compared with normal control group,CD8+T cells and CD4+Tregs in recurrent/refractory B-ALL patients before CAR-T cell infusion were increased,and the CD4/CD8 ratio was decreased.The proportion of CD4+Tregs at 1 week after CAR-T cell infusion was also higher than that of healthy controls,however,no significant difference was found between preinfusion and 1 week after infusion groups.Besides,compared with the non-remission(NR)group,the percentages of CD4+Tregs in complete remission were higher.CD4+Treg cell was chosen as the optimal T-cell subset for predicting the therapeutic efficacy of CAR-T cells by Receiver Operating Characteristic(ROC),and the AUC was 0.770(P=0.013).Patients with higher Tregs before infusion had shorter OS and RFS than patients with lower Tregs.The multivariate Cox proportional risk model showed that higher Tregs before CAR-T cell infusion was an independent adverse prognostic factor of RFS and OS.In addition,T cell subsets before infusion did not correlated with CRS.The proportions of CD3+T cells,CD8+T cells and CD4+Tregs before infusion were negatively related to CAR-T cell retention time(R=-0.673,-0.511,and-0.631;P=0.002,0.025 and 0.004).The optimal value for CD4+Tregs after infusion was 5.02% according to the ROC curve and AUC.Patients with higher Tregs after infusion had shorter OS and RFS than patients with lower Tregs.Multivariate Cox proportional risk model proved that higher Tregs after CAR-T cell infusion was an independent adverse prognostic factor of RFS and OS.Normal pre-lymphodepletion platelet count was associated with better RFS.Higher per-lymphodepletion LDH and active EMD before transfusion were independent adverse factors affecting RFS.In addition,CD4+Tregs after infusion did not correlated with CRS,but negatively related to CAR-T cell retention time(R=-0.579,P=0.019).Patients with hematopoietic stem cell transplantation(HSCT)after remission of CAR-T cell therapy had longer RFS and OS.There were differences in age and number of previous recurrence between HSCT group and non-HSCT group,but no differences in gender,tumor load,active extramedullary lesions,and the expression of CD4+Tregs before and after transfusion.Conclusion There were serious T cell immune dysfunction in recurrent/refractory B-ALL patients,especially abnormal elevation of CD4+Tregs.CD4+Tregs in recurrent/refractory B-ALL patients before and after infusion could be used to predict the prognosis and recurrence of CAR-T therapy.And Tregs after infusion was more valuable.Moreover,Tregs levels were not associated with cytokine release syndrome,but negatively correlated with the retention time of CAR-T cells in patients.HSCT after CAR-T therapy was beneficial to reduce recurrence of B-ALL and improve survival,and also may reverse the poor prognosis caused by abnormal elevation of Treg cells.In conclusion,we believed that abnormal elevation of CD4+Tregs in recurrent/refractory B-ALL patients affected the efficacy of CAR-T cell therapy,and may become an important target for further improving the efficacy of CAR-T cell therapy and reducing recurrence in the future.