DataSheet_1_The frequency of differentiated CD3+CD27-CD28- T cells predicts response to CART cell therapy in diffuse large B-cell lymphoma.docx

BackgroundChimeric antigen receptor T (CART) cell therapy targeting the B cell specific differentiation antigen CD19 has shown clinical efficacy in a subset of relapsed/refractory (r/r) diffuse large B cell lymphoma (DLBCL) patients. Despite this heterogeneous response, blood pre-infusion biomarkers predicting responsiveness to CART cell therapy are currently understudied.
MethodsBlood cell and serum markers, along with clinical data of DLBCL patients who were scheduled for CART cell therapy were evaluated to search for biomarkers predicting CART cell responsiveness.
FindingsCompared to healthy controls (n=24), DLBCL patients (n=33) showed significant lymphopenia, due to low CD3+CD4+ T helper and CD3-CD56+ NK cell counts, while cytotoxic CD3+CD8+ T cell counts were similar. Although lymphopenic, DLBCL patients had significantly more activated HLA-DR+ (P=0.005) blood T cells and a higher frequency of differentiated CD3+CD27-CD28- (28.7 ± 19.0% versus 6.6 ± 5.8%; P<0.001) T cells. Twenty-six patients were infused with CART cells (median 81 days after leukapheresis) and were analyzed for the overall response (OR) 3 months later. Univariate and multivariate regression analyses showed that low levels of differentiated CD3+CD27-CD28- T cells (23.3 ± 19.3% versus 35.1 ± 18.0%) were independently associated with OR. This association was even more pronounced when patients were stratified for complete remission (CR versus non-CR: 13.7 ± 11.7% versus 37.7 ± 17.4%, P=0.001). A cut-off value of ≤ 18% of CD3+CD27-CD28- T cells predicted CR at 12 months with high accuracy (P<0.001). In vitro, CD3+CD8+CD27-CD28- compared to CD3+CD8+CD27+CD28+ CART cells displayed similar CD19+ target cell-specific cytotoxicity, but were hypoproliferative and produced less cytotoxic cytokines (IFN-γ and TNF-α). CD3+CD8+ T cells outperformed CD3+CD4+ T cells 3- to 6-fold in terms of their ability to kill CD19+ target cells.
InterpretationLow frequency of differentiated CD3+CD27-CD28- T cells at leukapheresis represents a novel pre-infusion blood biomarker predicting a favorable response to CART cell treatment in r/r DLBCL patients.

背景：靶向B细胞特异性分化抗原CD19的嵌合抗原受体T（Chimeric Antigen Receptor T, CART）细胞疗法，在部分复发难治性（relapsed/refractory, r/r）弥漫大B细胞淋巴瘤（diffuse large B cell lymphoma, DLBCL）患者中展现出临床疗效。尽管该疗法应答存在异质性，目前针对可预测CART细胞疗法响应性的输注前血液生物标志物的研究仍较为匮乏。
方法：本研究对拟接受CART细胞疗法的DLBCL患者的血细胞、血清标志物及临床资料进行检测分析，以筛选可预测CART细胞疗法响应性的生物标志物。
结果：与健康对照组（n=24）相比，DLBCL患者（n=33）存在显著淋巴细胞减少，这与CD3+CD4+辅助性T细胞及CD3-CD56+自然杀伤（NK）细胞计数降低相关，而细胞毒性CD3+CD8+T细胞计数无显著差异。尽管存在淋巴细胞减少，DLBCL患者血液中活化型人类白细胞抗原-DR阳性（HLA-DR+，P=0.005）T细胞比例显著升高，且分化型CD3+CD27-CD28- T细胞占比更高（28.7±19.0% 相较于 6.6±5.8%；P<0.001）。共有26例患者接受了CART细胞输注（白细胞采集术后中位间隔81天），并于输注后3个月评估总缓解率（overall response, OR）。单因素及多因素回归分析显示，低水平的分化型CD3+CD27-CD28- T细胞（23.3±19.3% 相较于 35.1±18.0%）与总缓解率独立相关。当按完全缓解（complete remission, CR）分层分析时，该关联更为显著（CR组vs非CR组：13.7±11.7% 相较于 37.7±17.4%，P=0.001）。以CD3+CD27-CD28- T细胞占比≤18%作为截断值，可高精度预测12个月时的完全缓解（P<0.001）。体外实验显示，相较于CD3+CD8+CD27+CD28+ CART细胞，CD3+CD8+CD27-CD28- CART细胞对CD19+靶细胞的特异性细胞毒性水平相近，但增殖能力更低，且分泌的细胞毒性细胞因子（干扰素-γ（IFN-γ）与肿瘤坏死因子-α（TNF-α））更少。在杀伤CD19+靶细胞的能力方面，CD3+CD8+ T细胞的效果是CD3+CD4+ T细胞的3至6倍。
结论：白细胞采集术中分化型CD3+CD27-CD28- T细胞的低占比，可作为一种新型输注前血液生物标志物，用于预测复发难治性DLBCL患者对CART细胞治疗的良好应答。