Using The Flow Cytometry To Monitor And Screen The Quality Of The Starting T Cells Before CART Cell Preparation

Background Chimeric Antigen Receptor T Cells(CART Cells)therapy has achieved remarkable results in the treatment of malignant tumors,especially in hematological malignancies.The total remission rate(Overall Remission Rate,ORR)of targeting CD19 CART cells in the treatment of relapsed/refractory leukemia or lymphoma by various research centers at home and abroad can exceed 90%.So far,four CART cell products have been approved by the US Food and Drug Administration(Food and Drug Administration)for clinical treatment of various relapsed and refractory B cell tumors.The rapid development of CART cell therapy technology,with the accumulation of data,the problems it faces have gradually emerged.For example,patients lack the corresponding therapeutic targets or the disease progresses rapidly without time;or there are targets but cannot be prepared in vitro to obtain the minimum required amount of CART cells for treatment;even about 10-20% of patients even successfully prepare sufficient numbers CART cells,but after the infusion,the immune resistance appears,and the tumor cells in the body cannot be eliminated.There are many reasons for the above situation,one of the important factors is the "seed cells" used to prepare CART cells-that is,the number of starting T cells(The Starting T cells)is insufficient or the function is reduced.At present,the CART cell products approved by the US FDA for clinical treatment and most clinical trials are derived from peripheral blood mononuclear cells(PBMC)of patients.The disadvantages of autologous CART cells are: The conditions of each patient are different,so the number,composition and function of CART cells obtained are also very different;CART cell preparation process is complicated,the preparation cycle is long and the cost is expensive,etc.,and it cannot be successful every time preparation.In particular,the CART cell therapy effect of the initial T cell preparation provided by healthy donors has achieved good results,so the choice of autologous or allogeneic has also become a factor considered by researchers.There is no uniform prediction standard for which patients are suitable for autologous CART cell therapy.Objective Through the use of multi-parameter flow cytometry to detect and analyze the number,composition and function of the patient's the starting T cells and other related cell populations,to screen out indicators related to the success rate and efficiency of CART cell preparation,and establish a Rapid and accurate evaluation system,early prediction of the therapeutic effect of CART cells.Methods Taking r/r B-ALL patients who have been treated with CART cell therapy in our hospital since 2015 as the research object,multi-parameter flow cytometry was used to detect the number of the starting T cells,the proportion of each cell subgroup and tumor cells;The normal reference values of PD-1,PD-L1,perforin,and granzyme B on each subgroup of normal human T lymphocytes;establish an experimental method for cytokine profile detection using flow microsphere analysis(Cytometric Bead Assay,CBA)technology,and screen out For indicators related to the effect of CART cell therapy,develop a complete system for evaluating the number and function of starting T cells in order to screen out patients with high-risk factors that are not suitable for preparing CART cells from autologous starting T cells.The data used in this experiment was analyzed and processed by SPSS19.0 and Prism6.0 statistical software.Results1.The number of the initial T cells in CART cells was higher than that of the preparation failure group(1560/u L& 495/u L,P=0.033),the same CART cell therapeutic effective group start T cells was higher than the treatment invalid group(1600/u L & 610/u L,P=0.109)indicating that the number of start T cells is an important factor affecting CART cell therapy.2.CART cell preparation of the lymphocyte ratio of patients with successful group,the ratio of CD4/CD8 ratio and B lymphocyte is higher than the preparation failure group(44.80 ± 24.64 & 9.70 ± 1.06,P = 0.007;2.43 ± 1.04 & 0.57 ± 0.28,P = 0.000;4.22 ± 7.17 & 0.09 ± 0.18,P= 0.001);the proportion of regulatory T cells prepared for success group is lower than the preparation failure group(6.57 ± 3.58 & 11.02 ±3.53,P= 0.041),prompt The above indicators are related to the preparation of CART cells;and the regulatory T cells of CART cell therapy for effective groups are 5.38 ±2.52,and the treatment invalid group is 11.06±4.37,and there is statistical significance among the two groups(P= 0.004).It indicates that regulatory T cells are related to CART cell therapeutic effects.3.Perforin and Granzyme B can be detected in each subpopulation of healthy people T lymphocytes.As a result,the percentage of Perforin and Granzyme B on NK cells are highest.The confidence interval range is 76.56%(95% confidence intervals range from 63.24-89.87)and 71.32%(95% confidence intervals range from 52.38-90.26).CD3+CD8+ T cells was the second,the percentage of Perforin and Granzyme B on CD3+CD8 + T cells is 42.96,%(95% confidence interval ranges from 31.55-54.37)and 36.44%(95% confidence interval ranges from 23.71-49.17),respectively.Then it was NKT cells,the percentage of Perforin and Granzyme B on NKT cells is55.11%(95% confidence interval ranges from 40.97-69.26)and 38.27%(95%confidence interval ranges from 24.83-51.70);and the lowest is CD3+CD4+ T cells expressed,there are 39.08%(95% confidence interval ranges from 15.83-62.32)and9.77%(95% confidence interval range 6.77-12.56),respectively.These suggested that each subpopulation of T lymphocytes may play cells by Perforin/Granzyme B pathway Toxic effect.According to the biological characteristics of Perforin and Granzyme B,the lowest value of the Perforin and Granzyme B is less than 95%,which can be considered to be abnormally decreased by Perforin and Granzyme B expression,which in turn affects T lymphocytes.4.The detection of cytokinetic spectrum maturity by Cytometric Bead Array,has been detected in 100 cases of leukemia,lymphoma and normal human.The serum include IL-1?,IL-2,IL-4,IL-5,IL-6,IL-8,IL-10,IL-12P70,IL-17,TNF-?,IFN-?,IFN-?.Changes in cytokine spectrum in serum during blood collection can be used as one of the indicators reflecting the quality of the starting T cells.5.Flow cytometry detection of the expression of PD-1 and PD-L1 in different lymphocyte subpopulations of health people,and shows that the proportion of PD-1and PD-L1 on total T lymphocytes of health people is not high.The proportion of PD-1 on the CD3+ T cell(accounting for the ratio of CD3+ T cell ratio)is 2.96(95%confidence interval ranges from 1.92-4.00),and the ratio of PD-L1 is 0.79(95%confidence interval ranges from 0.30-1.28).So the upper limit of each indicator is higher than 95% in this study is considered to be out of normal range,the specific proportion of PD-1 in CD3+ T cells(account for CD3+ T cell ratio)> 4.00%;proportion of PD-1 on CD3+CD4+ T cells(CD3+CD4+T cell ratio)> 4.49%;CD3 +CD8 + T cell PD-1 ratio(account for CD3 + CD8 + T cell ratio)> 3.93%;proportion of PD-1 on Treg cells(Treg cells)> 1.69%;TEFF cells PD-1 ratio> 2.86%.We also established the method to detecte the memory T in different stages.The experimental method of cells provides a new monitoring index for screening the quality and function of the starting T cells.6.Use the existing experimental results to screen the indicators related to CART cell therapy,and establish an assessment system.At present,the main indicators included include: CD3+ T cells,lymphocyte ratio,CD4/CD8 ratio,Treg cell ratio,B lymphocyte proportion,the percentage of leukemia cells in peripheral blood,Granzyme B on CD3+CD8+ T cells,the proportion of B cells,the ratio of PD-1 and PD-L1 on CD3+CD8+ T cells and the change in cytokine spectrum when the starting T cells are collected.Other indicators such as memory T cells and myeloid source inhibitory cells are accumulating the number of cases.Whether it is incorporated into the assessment system in the future,further data support is still needed.Conclusion Multi-parameter flow cytometry is closely related to the number and quality monitoring and screening of the starting T cells,closely related to the preparation of CD19 CART cells,of which the number of start T cells,lymphocyte ratio,CD4 / CD8 ratio,B lymphocyte ratio and Treg ratio include important predictive value;and new indicators such as CD3+CD4-CD8-T cells in the cell population and TSCM ells,poroins and particles in each lymphocyte subpoplades.The ratio of Perforin and Granzyme B,the cytokine factor spectrum changes in serum during blood collection,the proportion of exhaustive T cells in each lymphocyte subpopulation also has preliminary significance,but it is not necessary to further verify its diagnostic value.