【作者】 孙婷；

【导师】 李登举； 周剑峰；

【作者基本信息】 华中科技大学 ， 内科学， 2024， 博士

【摘要】 目的:描述嵌合抗原受体T细胞(Chimeric antigen receptor T cell,CAR-T)治疗后,患者血液毒性的临床特征。评估自体造血干细胞移植(Autologous stem cell transplantation,ASCT)序贯CAR-T治疗方案的安全性。对比血液毒性严重程度不同的患者临床特征,寻找与血液毒性严重程度相关的临床因素。体外共培养实验,验证CAR-T杀伤靶细胞同时,损伤造血干祖细胞(Hematopoietic stem and progenitor cell,HSPC)功能的猜想。探索血液毒性的病理生理学机制,为后续寻找CAR-T治疗后血液毒性的干预措施提供依据。方法:本研究为单中心回顾性研究,纳入本中心于2019年至2022年3月接受CAR-T治疗的B细胞淋巴瘤患者,分为ASCT序贯CAR-T组和CAR-T组。主要结局指标为失访、死亡、疾病进展或复发、开始使用西达本胺或布鲁顿酪氨酸酶抑制剂维持治疗。依据不良事件通用术语标准(Common Terminology Criteria for Adverse Events,CTCAE)对患者CAR-T治疗后血液毒性严重程度进行分级。采用Fisher’s exact test、Kruskal-Wallis test、Mann-Whitney U test 和 Spearman correlation test 进行统计分析。通过体外共培养实验,结合流式细胞术,使用One-way ANOVA分析,比较不同共培养条件下,HSPC分化、凋亡与周期状态。结合单细胞转录组测序(Single-cell RNA sequencing,scRNA-seq)技术,鉴定细胞亚群,并使用单细胞调节网络推理与聚类(Single-Cell Regulatory Network Inference and Clustering,SCENIC)分析、对差异基因进行基因本体(Gene ontology,GO)富集,验证CAR-T杀伤靶细胞同时对HSPC的功能损伤,寻找与功能损伤相关的潜在机制。结果:共纳入164例患者,其中ASCT序贯CAR-T组83人,CAR-T组81人。两组患者回输CAR-T后血液毒性有相似的临床特征。在预处理化疗和回输CAR-T后的CRS反应期,两组患者都经历早期血细胞减少。早期的血液毒性,ASCT序贯CAR-T组较CAR-T组更严重。在回输CAR-T完成后的14天内,ASCT序贯CAR-T组达到CTCAE 3-4级的严重中性粒细胞减少发生率、严重血小板减少的发生率均达100%,而CAR-T组分别达到98%、54%。随着预处理化疗、CRS反应消退,两组患者的外周血细胞计数均逐渐恢复。ASCT序贯CAR-T组的早期严重血液毒性恢复更加迅速,严重中性粒细胞减少,中位在回输完成后第11天恢复,而CAR-T组中位在回输完成后第15天恢复(P<0.0001)。严重血小板减少,ASCT序贯CAR-T组中位在回输完成后第13天恢复,而CAR-T组中位在回输完成后第27天恢复(P=0.0089)。然而,早期的血液毒性恢复是不稳定的,两组患者,均随后高发晚期血细胞减少。在回输CAR-T完成的21天后,严重中性粒细胞减少在ASCT序贯CAR-T组和CAR-T组的发生率分别为79.52%、80.25%;严重血小板减少发生率分别为46.99%,CAR-T组可达50.62%,晚期中性粒细胞减少、血小板减少的严重程度,两组无显著性差异。ASCT序贯CAR-T组与CAR-T组治疗后,与晚期中性粒细胞减少和血小板减少相关的临床因素是类似的。基线时中性粒细胞绝对值(Absolute neutrophil count,ANC)、血小板计数(Platelet count,PLT)和血红蛋白浓度(Hemoglobulin concentration,Hb)与晚期ANC、PLT最低水平正相关。基线血清白介素6(Interleukin6,IL-6)浓度、回输后第一个月的血清铁蛋白浓度峰值、回输后第2月-第3月的血清乳酸脱氢酶(Lactate dehydrogenase,LDH)、C 反应蛋白(C-reactive protein,CRP)、铁蛋白、IL-6 和纤维蛋白原水平与晚期ANC、PLT最低水平负相关。体外共培养实验发现,CAR-T促进HSPC分化。将CD34阳选细胞与CAR-T直接共培养、间接共培养,与CAR-T杀伤Raji直接共培养、间接共培养后,CD34+CD38-亚群平均占CD34+亚群47.34%,62.55%,31.34%和41.33%,而空白对照组平均占70.67%,与对照共培养条件相比,CD34+CD38-亚群占比显著下降(P<0.0001)。CAR-T杀伤靶细胞同时,促进HSPC凋亡。与其他对照共培养条件相比,CD34+亚群、CD34+CD38-亚群以及CD34+CD38-CD45RA-亚群中的平均细胞凋亡比例都最高(平均细胞凋亡比例:CD34+亚群,16.70%;CD34+CD38-亚群,16.53%;CD34+CD38-CD45RA-亚群,12.75%),远高于其他共培养条件(P值:CD34+亚群,<0.0001;CD34+CD38-亚群,=0.0100;CD34+CD38-CD45RA-亚群,=0.0069)。CAR-T 杀伤靶细胞同时,促进HSPC周期停滞。以空白对照为参照,CAR-T杀伤Raji与CD34阳选细胞的直接共培养条件下,CD34+亚群、CD34+CD38-亚群以及CD34+CD38-CD45RA-亚群中的平均G0期细胞比例都显著升高,G0期细胞比例平均为空白参照的:CD34+亚群,2.014倍;CD34+CD38-亚群,2.633 倍;CD34+CD38-CD45RA-亚群,2.23 倍(P 值:CD34+亚群,=0.0116;CD34+CD38-亚群,<0.0001;CD34+CD38-CD45RA-亚群,<0.0001)。scRNA-seq细胞亚群分布显示,CAR-T杀伤Raji与人CD34阳选细胞直接共培养条件下,导致巨核-红细胞祖细胞占比升高,而造血干细胞(Hematopoietic stem cell,HSC)亚群、多谱系潜能祖细胞(Multilineageprogenitor,Multilin)、粒细胞祖细胞亚群缺失。信号通路富集分析显示,经CAR-T共培养处理后的CD34阳选细胞,IL-1、肿瘤坏死因子和干扰素通路上调。SCENIC转录因子分析证实,炎症相关通路转录因子(NFKBs、STATs和IRFs)上调而和干性维持、造血输出相关的转录因子(GATAs、CEBPs)下调。同时,比对差异表达基因发现,CAR-T杀伤靶细胞共培养处理后,HSC亚群、Multilin亚群的DNA损伤修复功能均显著受损。结论:CAR-T治疗后,血液毒性具有早期血细胞减少和晚期血细胞减少的“双相”特征。早期血液毒性,ASCT序贯CAR-T组发生率较CAR-T组更高,但恢复更快;晚期血液毒性,两组发生率无显著差异。患者自身的造血储备、炎症状态,是与晚期血液毒性密切相关的影响因素。体外共培养实验证明,CAR-T存在的情况下,促进HSPC分化、凋亡、G0期细胞周期停滞,且在CAR-T杀伤Raji的直接共培养条件下最显为著。scRNA-seq证明,CAR-T诱导HSPC迅速分化,且分化趋势偏向巨核-红细胞系,CAR-T杀伤Raji的直接共培养条件下,HSC亚群、Multilin亚群、粒细胞祖细胞亚群缺失。经共培养后,多种炎症相关信号通路、转录因子上调,而与干性维持、正常造血输出相关的转录因子下调,同时,DNA修复功能受损。以上特征符合HSC衰老的表现。第一部分:B细胞淋巴瘤CAR-T疗法的血液毒性临床特征目的:描述CAR-T治疗后,患者血液毒性的临床特征。评估ASCT序贯CAR-T治疗方案的安全性。对比血液毒性严重程度不同的患者临床特征,寻找与血液毒性严重程度相关的临床因素。方法:本研究为单中心回顾性研究,纳入本中心于2019年至2022年3月接受CAR-T治疗的B细胞淋巴瘤患者,分为ASCT序贯CAR-T组和CAR-T组。主要结局指标为失访、死亡、疾病进展或复发、开始使用西达本胺或布鲁顿酪氨酸酶抑制剂维持治疗。依据CTCAE对患者CAR-T治疗后血液毒性严重程度进行分级。采用Fisher’s exact test、Kruskal-Wallis test、Mann-Whitney U test 和 Spearman correlation test进行统计分析。结果:共纳入164例患者,其中ASCT序贯CAR-T组83人,CAR-T组81人。两组患者回输CAR-T后血液毒性有相似的临床特征。在预处理化疗和回输CAR-T后的CRS反应期,两组患者都经历早期血细胞减少。早期的血液毒性,ASCT序贯CAR-T组较CAR-T组更严重。在回输CAR-T完成后的14天内,ASCT序贯CAR-T组达到CTCAE 3-4级的严重中性粒细胞减少发生率、严重血小板减少的发生率均达100%,而CAR-T组分别达到98%、54%。随着预处理化疗、CRS反应消退,两组患者的外周血细胞计数均逐渐恢复。ASCT序贯CAR-T组的早期严重血液毒性恢复更加迅速,严重中性粒细胞减少,中位在回输完成后第11天恢复,而CAR-T组中位在回输完成后第15天恢复(P<0.0001)。严重血小板减少,ASCT序贯CAR-T组中位在回输完成后第13天恢复,而CAR-T组中位在回输完成后第27天恢复(P=0.0089)。然而,早期的血液毒性恢复是不稳定的,两组患者,均随后高发晚期血细胞减少。在回输CAR-T完成的21天后,严重中性粒细胞减少在ASCT序贯CAR-T组和CAR-T组的发生率分别为79.52%、80.25%;严重血小板减少发生率分别为46.99%,CAR-T组可达50.62%,晚期中性粒细胞减少、血小板减少的严重程度,两组无显著性差异。ASCT序贯CAR-T组与CAR-T组治疗后,与晚期中性粒细胞减少和血小板减少相关的临床因素是类似的。基线时ANC、PLT和Hb与晚期ANC、PLT最低水平正相关。基线IL-6浓度、回输后第一个月的血清铁蛋白浓度峰值、回输后第2月-第3月的血清LDH、CRP、铁蛋白、IL-6和纤维蛋白原水平与晚期ANC、PLT最低水平负相关。结论:CAR-T治疗后,血液毒性具有早期血细胞减少和晚期血细胞减少的“双相”特征。早期血液毒性,ASCT序贯CAR-T组发生率较CAR-T组更高,但恢复更快;晚期血液毒性,两组发生率无显著差异。患者自身的造血储备、炎症状态,是与晚期血液毒性密切相关的影响因素。第二部分:B细胞淋巴瘤CAR-T疗法对造血干祖细胞功能影响的机制探索目的:观察B细胞淋巴瘤CAR-T疗法对HSPC功能影响,并探索其机制方法:通过体外共培养实验,结合流式细胞术,使用One-way ANOVA分析,比较不同共培养条件下,HSPC分化、凋亡与周期状态。结合scRNA-seq技术,鉴定细胞亚群,并使用SCENIC分析、对差异基因进行GO通路富集分析,验证CAR-T杀伤靶细胞同时对HSPC的功能损伤,寻找与功能损伤相关的潜在机制。结果:体外共培养实验发现,CAR-T促进HSPC分化。将CD34阳选细胞与CAR-T直接共培养、间接共培养,与CAR-T杀伤Raji直接共培养、间接共培养后,CD34+CD38-亚群平均占 CD34+亚群 47.34%,62.55%,31.34%和 41.33%,而空白对照组平均占70.67%,与对照共培养条件相比,CD34+CD38-亚群占比显著下降(P<0.0001)。CAR-T杀伤靶细胞同时,促进HSPC凋亡。与其他对照共培养条件相比,CD34+亚群、CD34+CD38-亚群以及CD34+CD38-CD45RA-亚群中的平均细胞凋亡比例都最高(平均细胞凋亡比例:CD34+亚群,16.70%;CD34+CD38-亚群,16.53%;CD34+CD38-CD45RA-亚群,12.75%),远高于其他共培养条件(P值:CD34+亚群,<0.0001;CD34+CD38-亚群,=0.0100;CD34+CD38-CD45RA-亚群,=0.0069)。CAR-T杀伤靶细胞同时,促进HSPC周期停滞。以空白对照为参照,CAR-T杀伤Raji与CD34阳选细胞的直接共培养条件下,CD34+亚群、CD34+CD38-亚群以及CD34+CD38-CD45RA-亚群中的平均G0期细胞比例都显著升高,G0期细胞比例平均为空白参照的:CD34+亚群,2.014 倍;CD34+CD38-亚群,2.633 倍;CD34+CD38-CD45RA-亚群,2.23 倍(P值:CD34+亚群,=0.0116;CD34+CD38-亚群,<0.0001;CD34+CD38-CD45RA-亚群,<0.0001)。scRNA-seq细胞亚群分布显示,CAR-T杀伤Raji与CD34阳选细胞直接共培养条件下,导致巨核-红细胞祖细胞占比升高,而HSC亚群、Multilin亚群、粒细胞祖细胞亚群缺失。信号通路富集分析显示,经CAR-T共培养处理后的CD34阳选细胞,IL-1、肿瘤坏死因子和干扰素通路上调。SCENIC转录因子分析证实,炎症相关通路转录因子(NFKBs、STATs和IRFs)上调而和干性维持、造血输出相关的转录因子(GATAs、CEBPs)下调。同时,比对差异表达基因发现,CAR-T杀伤靶细胞共培养处理后,HSC亚群、MultiLin亚群的DNA损伤修复功能均显著受损。结论:体外共培养实验证明,CAR-T存在的情况下,促进HSPC分化、凋亡、G0期细胞周期停滞,且在CAR-T杀伤Raji的直接共培养条件下最显为著。ScRNA-seq 证明,CAR-T 诱导 HSPC 迅速分化,且分化趋势偏向巨核-红细胞系,CAR-T 杀伤Raji的直接共培养条件下,HSC亚群、Multilin亚群、粒细胞祖细胞亚群缺失。经共培养后,多种炎症相关信号通路、转录因子上调,而与干性维持、正常造血输出相关的转录因子下调,同时,DNA修复功能受损。以上特征符合HSPC衰老的表现。更多还原

【Abstract】 Objective:Describing the clinical characteristics of hematotoxicity in patients receiving CAR-T therapy.Comparisons of the hematopoietic recovery process of patients after the two subgroups,ASCT-CAR-T and CAR-T,were made to evaluate the hematological safety of ASCT-CAR-T therapy.By comparing the clinical characteristics of patients with and without severe hematotoxicity,clinical factors related to the severity of hematotoxicity were highlighted.Moreover,in vitro co-culture experiments were carried out in order to verify the hypothesis that CAR-T therapy might damage the function of hematopoietic stem and progenitor cell(HSPC),which might be a potential pathophysiological mechanism of hematotoxicity,and might provide a basis for subsequent interventions for hematotoxicity after CAR-T therapy.Methods:The first part of the research was a single-center retrospective study.Patients who received ASCT-CAR-T therapy and classical CAR-T therapy at our center from 2019 to March 2022 were included.The primary end point was loss of follow-up,death,disease progression or recurrence,and the initiation of maintenance therapy with Chidamide or Bruton’s tyrosinase inhibitors.The severity of hematotoxicity after CAR-T therapy was graded according to the Common Terminology Criteria for Adverse Events(CTCAE).Fisher’s exact test,Kruskal-Wallis test,Mann-Whitney Utest and Spearman correlation test were used for statistical analysis.The second part of the research was a tentative exploration of the potential mechanism underlying CAR-T hematotoxicity.Through in vitro co-culture experiment,combined with flow cytometry,One-way ANOVA analysis was used to compare the differentiation,apoptosis and cycle status of HSPC under different co-culture conditions.With single-cell RNA sequencing(scRNA-seq)technology,cell subsets were identified,and single-cell regulatory network inference and clustering(SCENIC)analysis and gene ontology(GO)enrichment of differentially expressed genes were used to verify the functional damage of HSPCs,and to find the potential mechanisms related to functional damage.Results:In the first part of this research,a total of 164 patients were included,including 83 in the ASCT-CAR-T group and 81 in the classical CAR-T treatment group.The two groups of patients had similar clinical characteristics of hematotoxicity after CAR-T reinfusion.During the conditioning chemotherapy and CRS period,the two groups of patients experienced early-phase cytopenia.The early-phase hematotoxicity was more severe in the ASCT-CAR-T group than in the CAR-T group.Within 14 days after the completion of CART reinfusion,the incidence of severe neutropenia and severe thrombocytopenia reached CTCAE grade 3-4 in the ASCT-CAR-T group were both 100%,while those in the CAR-T group were 98%and 54%,respectively.With the regression of conditioning chemotherapy and CRS,the peripheral blood cell counts of the two groups of patients gradually recovered.The early-phase severe hematotoxicity of the ASCT-CAR-T group recovered more rapidly,with severe neutropenia recovering on the 11th day after the completion of CAR-T reinfusion,while the CAR-T group recovered on the 15th day after the completion of CAR-T reinfusion(P<0.0001).The median recovery time of severe thrombocytopenia in the ASCT-CAR-T group was 13 days after the completion of CAR-T reinfusion,while that in the CAR-T group was 27 days after the completion of CAR-T reinfusion(P=0.0089).Both groups had a high incidence of late-phase cytopenia.Twenty-one days after the completion of CAR-T reinfusion,the incidence of severe neutropenia in the ASCT-CAR-T group and the CAR-T group was 79.52%and 80.25%,respectively;the incidence of severe thrombocytopenia was 46.99%and 50.62%in the ASCT-CAR-T group,respectively.There was no significant difference in the severity of late-phase neutropenia and thrombocytopenia between the two groups.After treatment with ASCT-CAR-T and CAR-T,the clinical factors associated with late-stage neutropenia and thrombocytopenia were similar.The absolute neutrophil count(ANC),platelet count(PLT)and hemoglobulin concentration(Hb)at baseline were positively correlated with the nadir of ANC and PLT at the late-phase.The levels of LDH,CRP,ferritin,IL-6 and fibrinogen at 2 and 3 months after reinfusion were negatively correlated with the nadir of ANC and PLT at the late-phase.The second part of this research revealed that CAR-T promoted the differentiation of HSPC.After direct and indirect co-culture with CAR-T,or direct and indirect co-culture with CAR-T and Raji,the proportion of CD34+CD38-subgroup accounted for 47.34%,62.55%,31.34%and 41.33%of the CD34+subgroup,while the blank control group accounted for 70.67%,respectively.Compared with the control co-culture condition,the proportion of CD34+CD38-subgroup was significantly decreased(P<0.0001).HSPC apoptosis was promoted when cultured directly with CAR-T and Raji.Compared with other control coculture conditions,the average apoptosis rate of CD34+subpopulation,CD34+CD38subpopulation and CD34+CD38-CD45RA-subpopulation was the highest(average apoptosis rate:CD34+subpopulation,16.70%;CD34+CD38-subpopulation,16.53%;CD34+CD38-CD45RA-subpopulation,12.75%),and was much higher than other co-culture conditions(P value:CD34+subpopulation,<0.0001;CD34+CD38-subpopulation,=0.0100;CD34+CD38-CD45RA-subpopulation,=0.0069).G0-phase cell cycle arrest was also the highest in direct culture condition with CAR-T and Raji.Taking blank control as reference,the average G0 phase cell proportion of CD34+subpopulation,CD34+CD3 8-subpopulation and CD34+CD38-CD45RA-subpopulation was significantly increased,the average G0 phase cell proportion was 2.014 times of blank reference:CD34+subpopulation,2.634 times;CD34+CD38-subpopulation,2.633 times;CD34+CD38-CD45RA-subpopulation,2.23 times(P value:CD34+subpopulation,=0.0116;CD34+CD38-subpopulation,<0.0001;CD34+CD38-CD45RA-subpopulation,<0.0001).With scRNA-seq,cell subpopulation distribution showed that direct co-culture condition with CAR-T and Raji led to the increase of megakaryocyte-erythroid progenitors(MKPs),and the loss of HSC subpopulation,multilineage progenitor(Multilin)subpopulation and granulocyte progenitor subpopulation.GO pathway enrichment analysis showed that IL-1,tumor necrosis factor and interferon pathways were up-regulated after CAR-T co-culture treatment.SCENIC analysis confirmed that inflammation-related transcription factors(NFKBs,STATs and IRFs)were upregulated and stemness maintenance and hematopoietic output-related transcription factors(GATAs,CEBPs)were down-regulate simultaneously,pathway enrichment analysis showed that the DNA damage repair function of HSC subpopulation and Multilin subpopulation were significantly impaired after co-culture treatment of CAR-T and Raji.Conclusions:Hematotoxicity following ASCT-CAR-T therapy and classical CAR-T therapy both has the "biphasic" characteristics,which means that the patients are likely to experience a late-phase of cytopenia after previous recovery of an early-phase cytopenia happening immediately after CAR-T infusion.The incidence of severe cytopenia after ASCT-CAR-T therapy is higher in the early phase than after classical CAR-T therapy,but the recovery is faster;the incidence of late-phase hematotoxicity is not significantly different between the two groups.The hematopoietic reserve and inflammatory state of the patient are closely related to the late stage of hematological toxicity.In vitro co-culture experiments showed that the presence of CAR-T promoted the differentiation,apoptosis and G0 phase cell cycle arrest of HSPCs,which was most obvious under the direct co-culture condition of CAR-T and Raji.scRNA-seq showed that CAR-T induced HSPC to differentiate rapidly,and the differentiation trend was biased to megakaryocyte-erythroid lineage.Under the direct co-culture condition of CAR-T and Raji,the loss of HSC subpopulation,MultiLin cell subpopulation and granulocyte progenitor subpopulation was also detected.After co-culture,a variety of inflammation-related signaling pathways and transcription factors were up-regulated,while the transcription factors related to the maintenance of stemness and normal hematopoietic output were downregulated,and the DNA repair function was impaired.The above characteristics are consistent with the aging of HSPCs.Part Ⅰ:Clinical features of hematotoxicity in patients with B-cell lymphoma after CAR-T therapyObjective:Describing the clinical characteristics of hematotoxicity in patients receiving CAR-T therapy.Comparisons of the hematopoietic recovery process of patients after the two subgroups,ASCT-CAR-T and CAR-T,were made to evaluate the hematological safety of ASCT-CAR-T therapy.By comparing the clinical characteristics of patients with and without severe hematotoxicity,clinical factors related to the severity of hematotoxicity were highlighted.Methods:The first part of the research was a single-center retrospective study.Patients who received ASCT-CAR-T therapy and classical CAR-T therapy at our center from 2019 to March 2022 were included.The primary end point was loss of follow-up,death,disease progression or recurrence,and the initiation of maintenance therapy with Chidamide or Bruton’s tyrosinase inhibitors.The severity of hematotoxicity after CAR-T therapy was graded according to the CTCAE.Fisher’s exact test,Kruskal-Wallis test,Mann-Whitney U test and Spearman correlation test were used for statistical analysis.Results:In the first part of this research,a total of 164 patients were included,including 83 in the ASCT-CAR-T group and 81 in the classical CAR-T treatment group.The two groups of patients had similar clinical characteristics of hematotoxicity after CAR-T reinfusion.During the conditioning chemotherapy and CRS period,the two groups of patients experienced early-phase cytopenia.The early-phase hematotoxicity was more severe in the ASCT-CAR-T group than in the CAR-T group.Within 14 days after the completion of CART reinfusion,the incidence of severe neutropenia and severe thrombocytopenia reached CTCAE grade 3-4 in the ASCT-CAR-T group were both 100%,while those in the CAR-T group were 98%and 54%,respectively.With the regression of conditioning chemotherapy and CRS,the peripheral blood cell counts of the two groups of patients gradually recovered.The early-phase severe hematotoxicity of the ASCT-CAR-T group recovered more rapidly,with severe neutropenia recovering on the 11th day after the completion of CAR-T reinfusion,while the CAR-T group recovered on the 15th day after the completion of CAR-T reinfusion(P<0.0001).The median recovery time of severe thrombocytopenia in the ASCT-CAR-T group was 13 days after the completion of CAR-T reinfusion,while that in the CAR-T group was 27 days after the completion of CAR-T reinfusion(P=0.0089).Both groups had a high incidence of late-phase cytopenia.Twenty-one days after the completion of CAR-T reinfusion,the incidence of severe neutropenia in the ASCT-CAR-T group and the CAR-T group was 79.52%and 80.25%,respectively;the incidence of severe thrombocytopenia was 46.99%and 50.62%in the ASCT-CAR-T group,respectively.There was no significant difference in the severity of late-phase neutropenia and thrombocytopenia between the two groups.After treatment with ASCT-CAR-T and CAR-T,the clinical factors associated with late-stage neutropenia and thrombocytopenia were similar.The ANC,PLT and Hb at baseline were positively correlated with the nadir of ANC and PLT at the late-phase.The levels of LDH,CRP,ferritin,IL-6 and fibrinogen at 2 and 3 months after reinfusion were negatively correlated with the nadir of ANC and PLT at the late-phase.Conclusions:Hematotoxicity following ASCT-CAR-T therapy and classical CAR-T therapy both has the "biphasic" characteristics,which means that the patients are likely to experience a late-phase of cytopenia after previous recovery of an early-phase cytopenia happening immediately after CAR-T infusion.The incidence of severe cytopenia after ASCT-CAR-T therapy is higher in the early phase than after classical CAR-T therapy,but the recovery is faster;the incidence of late-phase hematotoxicity is not significantly different between the two groups.The hematopoietic reserve and inflammatory state of the patient are closely related to the late stage of hematological toxicity.Part Ⅱ:Mechanism exploration of the effect of CAR-T cells on hematopoietic stem and progenitor cell function in B-cell lymphomaObjective:In vitro co-culture experiments were carried out in order to verify the hypothesis that CAR-T therapy might damage the function of hematopoietic stem and progenitor cell(HSPC),which might be a potential pathophysiological mechanism of hematotoxicity,and might provide a basis for subsequent interventions for hematotoxicity after CAR-T therapy.Methods:The second part of the research was a tentative exploration of the potential mechanism underlying CAR-T hematotoxicity.Through in vitro co-culture experiment,combined with flow cytometry,One-way ANOVA analysis was used to compare the differentiation,apoptosis and cycle status of HSPC under different co-culture conditions.With scRNA-seq technology,cell subsets were identified,and SCENIC analysis and GO enrichment of differentially expressed genes were used to verify the functional damage of HSPCs,and to find the potential mechanisms related to functional damage.RESULTS:The second part of this research revealed that CAR-T promoted the differentiation of HSPC.After direct and indirect co-culture with CAR-T,or direct and indirect co-culture with CAR-T and Raji,the proportion of CD34+CD38-subgroup accounted for 47.34%,62.55%,31.34%and 41.33%of the CD34+subgroup,while the blank control group accounted for 70.67%,respectively.Compared with the control coculture condition,the proportion of CD34+CD38-subgroup was significantly decreased(P<0.0001).HSPC apoptosis was promoted when cultured directly with CAR-T and Raji.Compared with other control co-culture conditions,the average apoptosis rate of CD34+subpopulation,CD34+CD3 8-subpopulation and CD34+CD38-CD45RA-subpopulation was the highest(average apoptosis rate:CD34+subpopulation,16.70%;CD34+CD38subpopulation,16.53%;CD34+CD38-CD45RA subpopulation,12.75%),and was much higher than other co-culture conditions(P value:CD34+subpopulation,<0.0001;CD34+CD38-subpopulation,=0.0100;CD34+CD38-CD45RA-subpopulation,=0.0069).G0phase cell cycle arrest was also the highest in direct culture condition with CAR-T and Raji.Taking blank control as reference,the average G0 phase cell proportion of CD34+subpopulation,CD34+CD3 8-subpopulation and CD34+CD38-CD45RA-subpopulation was significantly increased,the average G0 phase cell proportion was 2.014 times of blank reference:CD34+subpopulation,2.634 times;CD34+CD38-subpopulation,2.633 times;CD34+CD38-CD45RA-subpopulation,2.23 times(P value:CD34+subpopulation,=0.0116;CD34+CD38-subpopulation,<0.0001;CD34+CD38-CD45RA-subpopulation,<0.0001).With scRNA-seq,cell subpopulation distribution showed that direct co-culture condition with CAR-T and Raji led to the increase of MKPs,and the loss of HSC subpopulation,Multilin subpopulation and granulocyte progenitor subpopulation.GO pathway enrichment analysis showed that IL-1,tumor necrosis factor and interferon pathways were up-regulated after CAR-T co-culture treatment.SCENIC analysis confirmed that inflammation-related transcription factors(NFKBs,STATs and IRFs)were upregulated and stemness maintenance and hematopoietic output-related transcription factors(GATAs,CEBPs)were down-regulate simultaneously,pathway enrichment analysis showed that the DNA damage repair function of HSC subpopulation and Multilin subpopulation were significantly impaired after co-culture treatment of CAR-T and Raji.Conclusions:In vitro co-culture experiments showed that the presence of CAR-T promoted the differentiation,apoptosis and G0 phase cell cycle arrest of HSPCs,which was most obvious under the direct co-culture condition of CAR-T and Raji.scRNA-seq showed that CAR-T induced HSPC to differentiate rapidly,and the differentiation trend was biased to megakaryocyte-erythroid lineage.Under the direct co-culture condition of CAR-T and Raji,the loss of HSC subpopulation,MultiLin cell subpopulation and granulocyte progenitor subpopulation was also detected.After co-culture,a variety of inflammation-related signaling pathways and transcription factors were up-regulated,while the transcription factors related to the maintenance of stemness and normal hematopoietic output were downregulated,and the DNA repair function was impaired.The above characteristics are consistent with the aging of HSPCs.更多还原