背景与目的

在利妥昔单抗联合化疗的标准治疗模式下，仍有30-40%弥漫大B细胞淋巴瘤（DLBCL）患者因原发/继发耐药出现疾病进展或复发。嵌合抗原受体T（CAR-T）细胞治疗作为新兴的免疫疗法，为复发/难治DLBCL（R/R DLBCL）患者带来了治愈希望。早期复发患者通常对挽救化疗不敏感，更可能从CAR-T治疗中获益，故而早期识别此类CAR-T获益人群尤其重要。此外，R/R DLBCL患者接受CAR-T治疗后虽然近50%患者可获得长期缓解，但仍有部分患者无法获得缓解或出现早期复发。CAR-T治疗疗效的分子预测体系尚未完全建立，关于患者自身T细胞功能与CAR-T治疗疗效，尤其是长期疗效的相关机制亟待阐明。基于以上背景，我们将从以下几方面展开研究：1）探究利妥昔单抗联合化疗标准方案下DLBCL患者早期复发的危险因素，更好地识别能从CAR-T中获益的高危患者；2）借助单细胞测序探索接受CAR-T治疗后获得长期缓解患者自身免疫重建情况；3）探索DLBCL患者自身T细胞DNA甲基化图谱变化特征与CAR-T疗效相关性。

方法

回顾性分析2009年1月至2017年5月在北京协和医院确诊的564例初治DLBCL患者经一线标准治疗后出现早期复发患者的临床特征，分析早期复发的危险因素及生存预后。

纳入5例经CAR-T治疗后实现持续性完全缓解的复发/难治弥漫大B细胞淋巴瘤患者，留取患者清淋预处理前（基线）、CAR-T细胞回输后第14天（D14）、CAR-T细胞回输后D28、治疗后长期随访（回输后1年左右）的外周血标本送检单细胞转录组测序及T细胞受体（TCR）测序，揭示CAR-T治疗后免疫系统时序性动态演变规律，并深度解析免疫细胞亚群的功能表型动态调控特征。

纳入18例接受CAR-T细胞治疗的复发/难治弥漫大B细胞淋巴瘤患者。在接受CAR-T治疗前及回输CAR-T细胞后D28评估疗效时收集患者外周血样本，分选CD3+T细胞并进行MethylationEPIC(850K) Bead Chip或MethylationEPIC(935K) Bead Chip检测（芯片迭代）。分析CAR-T治疗后获完全缓解（CR）和未获得CR患者（Non-CR）治疗前后差异甲基化位点（DMP），并进行GO、KEGG富集分析。

结果

564例初治DLBCL患者在接受利妥昔单抗联合化疗一线标准方案治疗后，413例患者（73.2%）获得了CR。中位随访时间为65.1个月，59例患者出现复发，其中32例（54.2%）在1年内复发，27例（45.8%）在CR后1年或以上复发。出现早期复发的患者相较于在结疗1年后出现复发的患者，具有更高的国际预后指数（IPI）评分（P=0.004）和东部肿瘤协作组（ECOG）评分（P=0.02），且乳酸脱氢酶（LDH）更高（P=0.002）。其中，ECOG评分和IPI评分在多因素分析中仍存在统计学意义，两者P值分别为0.018和0.039，相对危险度（OR）分别为0.274和0.227。1年后复发的患者比1年内复发的患者OS更长（中位OS 42.65月vs 24.2月；P=0.04）。对OS的多因素分析显示，1年内复发是影响生存的独立预后参数，风险比（HR）为 0.24（P=0.002）。

5例接受CAR-T治疗获得长期缓解的复发/难治DLBCL患者在CAR-T治疗后免疫细胞亚群发生了显著变化。我们发现，效应CD8 T细胞在CAR-T回输后D14、D28细胞比例及毒性评分升高，后期出现回落，但仍高于基线水平。调节性T细胞（Treg细胞）在CAR-T细胞回输后D14 细胞比例较基线明显升高，回输后D28迅速回落。在CAR-T回输后长期随访标本中，Treg细胞比例较基线显著减低并存在统计学差异。进一步纵向比较CAR-T治疗不同时间点的差异表达基因并进行富集分析，我们发现，效应CD8 T细胞在CAR-T回输后D14较基线上调表达的差异基因主要富集在T细胞激活、趋化因子生成、细胞毒性等通路；在CAR-T回输后D28，相较于回输后D14，上调基因主要富集在抗病毒反应、I类干扰素信号通路及IFN-γ介导信号通路，下调差异基因富集在细胞因子分泌正向调节通路。在长期随访标本中，与效应CD8 T细胞激活功能相关的生物学过程及通路如T细胞激活通路等出现明显下调。Treg细胞在CAR-T细胞治疗中具有双相功能表型，在CAR-T治疗早期（回输后D14）呈现功能性激活，与基线相比的差异基因主要富集在T细胞激活、IKK/NF-κB信号转导通路，限制过度炎症反应，维持机体免疫稳态；在治疗后期（回输后D28），差异基因则富集在凋亡、I类干扰素等通路，呈现耗竭表型。

18例接受CAR-T治疗的复发/难治DLBCL患者，中位随访10.3月，总生存率为77.8%。其中，完全缓解（CR）组12例，非完全缓解（Non-CR）组6例。我们发现，虽然CR组和Non-CR组治疗前后整体甲基化水平均无明显差异，但是两组在治疗前后甲基化景观都发生了显著的动态变化。在CR组，治疗前后的差异甲基化位点KEGG富集分析主要富集在细胞因子通路、趋化因子通路、Th1/2分化通路等。在Non-CR组，差异甲基化位点主要富集在轴突导向、Rap1信号通路、PI3K-Akt信号通路。将CAR-T细胞回输后CR组和Non-CR组的DNA甲基化位点进一步行KEGG富集分析发现，CR组相较于Non-CR组，在Rap1信号通路、T细胞受体通路、PI3K-Akt等通路上呈现明显富集，且呈现上调趋势。通过对比基线和CAR-T治疗后两个时间点CR组和Non-CR组组间T细胞DNA甲基化差异最大且具有注释基因的前20位差异甲基化位点，我们发现有8个基因在基线两组间存在甲基化差异，且不随治疗变化，它们分别是PAX8，AMD1，SORCS2，PAX8-AS1，TMEM219，EPHA2，GSTM1和LIPT2。

结论

ECOG和IPI评分是DLBCL 1年内复发的独立危险因素，1年内复发的DLBCL患者预后更差，并且早期复发是复发患者生存的独立预后因素。

效应CD8 T细胞在CAR-T回输后D14、D28均具有良好免疫应答，后期效应CD8 T细胞恢复稳态，但较治疗前具有更强的免疫应答能力。Treg细胞在CAR-T细胞治疗中具有双相功能表型，在CAR-T治疗早期呈现功能性激活，限制过度炎症反应；在治疗后期呈现耗竭表型，解除机体免疫抑制，利于疾病长期缓解。

CAR-T治疗对患者自身T细胞具有表观遗传学重塑效应，并可能由此影响T淋巴细胞的功能，在适应性免疫反应中发挥了重要作用。CR组与Non-CR组DNA甲基化图谱具有明显差异，我们推测有8个基因的甲基化状态可能具有疗效预测价值。

Objectives: Despite the standard treatment regimen of rituximab combined with chemotherapy, 30-40% of diffuse large B-cell lymphoma (DLBCL) patients eventually experience disease progression or relapse, primarily due to primary or secondary resistance. Chimeric Antigen Receptor T (CAR-T) cell therapy, as an emerging immunotherapy, has brought hope for a cure in patients with relapsed/refractory DLBCL (R/R DLBCL). Early relapsed patients are often less sensitive to salvage chemotherapy and are more likely to benefit from CAR-T therapy, highlighting the importance of early identification of patients who are most likely to benefit from this treatment. Moreover, although nearly 50% of R/R DLBCL patients achieve long-term remission after CAR-T treatment, some patients fail to achieve remission or experience early relapse. A molecular prediction system for CAR-T treatment efficacy is yet to be fully established, and there is an urgent need to investigate the mechanisms related to intrinsic T cell function and the long-term efficacy of CAR-T therapy. In this context, our research will focus on the following aspects: 1) Investigating the risk factors for early relapse in DLBCL patients treated with standard rituximab plus chemotherapy to better identify high-risk patients who could benefit from CAR-T therapy; 2) Characterizing immune reconstitution in patients with durable remission following CAR-T therapy through single-cell RNA sequencing; 3) Elucidating the correlation between DNA methylation remolding of patients' intrinsic T cells and the efficacy of CAR-T therapy.

Methods: 1. A retrospective analysis was conducted on the clinical characteristics, risk factors of early relapse, and survival outcomes of 564 newly diagnosed DLBCL patients at Peking Union Medical College Hospital from January 2009 to May 2017 who experienced early relapse after first-line standard treatment.  2. Five patients with R/R DLBCL achieving sustained complete remission after CAR-T therapy were included. Longitudinal peripheral blood samples were collected at four critical phases: pre-lymphodepletion (baseline), day 14 (D14), day 28 (D28) post-CAR-T cell infusion, and during long-term follow-up (1 year after infusion). These samples underwent single-cell transcriptomic and T-cell receptor (TCR) sequencing to systematically characterize the immune reconstitution dynamics and functional phenotypic evolution of immune cell subsets following CAR-T intervention.3. Eighteen patients with R/R DLBCL patients undergoing CAR-T cell therapy were enrolled. Peripheral blood samples were collected pre-treatment and at D28 post-infusion. CD3+ T cells were sorted, and MethylationEPIC (850K) Bead Chip or MethylationEPIC (935K) Bead Chip detection (chip iteration) was performed. Differentially methylated positions (DMPs) between patients who achieved complete remission (CR) and those who did not (Non-CR) were analyzed before and after treatment, followed by GO and KEGG enrichment analysis.

Results: 1. Among 564 newly diagnosed DLBCL patients receiving first-line rituximab-based chemotherapy, 413 patients (73.2%) achieved CR. After a median follow-up of 65.1 months, 59 patients relapsed, stratified into early (< 1 year post-CR, n=32, 54.2%) and late (≥1 year post-CR, n=27, 45.8%) relapse cohorts. Patients with early relapse showed higher International Prognostic Index (IPI) scores (P=0.004), Eastern Cooperative Oncology Group (ECOG) scores (P=0.02), and lactate dehydrogenase (LDH) levels (P=0.002) compared to those who relapsed later than 1 year. Multivariate analysis identified ECOG and IPI scores as independent predictors of early relapse, with P-values of 0.018 and 0.039, respectively, and odds ratios (OR) of 0.274 and 0.227. Patients who relapsed after 1 year had significantly better overall survival (OS) than those who relapsed within 1 year (median OS: 42.65 months vs. 24.2 months; P=0.04). Multivariate analysis of OS indicated that relapse within 1 year was an independent prognostic factor (HR 0.24; P=0.002). 2. In the cohort of five patients with relapsed/refractory DLBCL who achieved long-term remission after CAR-T therapy, significant changes were observed in immune cell subpopulations post-treatment. We found that effector CD8 T cells had an increased proportion and toxicity scores at both D14 and D28 post-CAR-T infusion, followed by a decline, although still higher than baseline levels. Regulatory T cells (Tregs) increased significantly at D14 but declined rapidly by D28, with their proportion significantly lower than baseline during long-term follow-up. Gene expression analysis of effector CD8 T cells revealed pathway enrichment in T cell activation, chemokine production, and cytotoxicity at D14. At D28 post-infusion, compared to D14, upregulated genes were primarily enriched in antiviral response, type I interferon signaling, and IFN-γ-mediated signaling pathways, while downregulated genes were enriched in pathways related to cytokine secretion regulation. In long-term follow-up samples, biological processes and pathways related to effector CD8 T cell activation, such as T cell activation pathways, showed significant downregulation. Treg cells exhibited a biphasic functional phenotype during CAR-T cell therapy. In the early treatment phase (Day 14 post-infusion), they demonstrate functional activation, with differentially expressed genes (DEGs) compared to baseline being significantly enriched in T-cell activation and IKK/NF-κB signaling pathways, limiting excessive inflammatory responses and maintaining immune homeostasis. In the late treatment phase (Day 28 post-infusion), DEGs are enriched in apoptosis and Type I interferon signaling pathways, reflecting an exhausted phenotype. 3. In the cohort of eighteen patients with R/R DLBCL who underwent CAR-T therapy, the median follow-up was 10.3 months, with an overall survival rate of 77.8%. Twelve patients achieved complete remission (CR), and six patients did not (Non-CR). We found that DNA methylation sequencing of T cells from both groups showed dynamic changes in the methylation landscape, although no significant overall difference in methylation levels between the CR and Non-CR groups. In the CR group, KEGG enrichment analysis of differentially methylated positions (DMPs) before and after treatment showed enrichment in cytokine signaling, chemokine signaling, and Th1/2 differentiation pathways. In the Non-CR group, DMPs were mainly enriched in axon guidance, Rap1 signaling, and PI3K-Akt signaling pathways. Further enrichment analysis of DNA methylation sites after CAR-T infusion revealed significant enrichment in the Rap1 signaling pathway, T cell receptor pathway, and PI3K-Akt pathway in the CR group compared to the Non-CR group, with an upregulation trend. Comparing the top 20 DMPs with annotated genes between baseline and post-CAR-T treatment in both groups, we found 8 genes with methylation differences at baseline, which did not change after treatment. These genes included PAX8, AMD1, SORCS2, PAX8-AS1, TMEM219, EPHA2, GSTM1, and LIPT2.

Conclusions: 1. ECOG and IPI scores are independent risk factors for early relapse in DLBCL, and early relapse is associated with a poorer prognosis. Early relapse is an independent prognostic factor for survival in relapsed patients. 2. Effector CD8 T cells show robust immune responses at D14 and D28 post-CAR-T infusion, returning to a steady state with improved immune response capability in the long term. Tregs exhibit a biphasic functional phenotype, with early activation to control inflammation and late exhaustion, contributing to long-term remission. 3. CAR-T therapy induces methylation remodeling of the patient’s T cells, impacting T lymphocyte function and playing a key role in adaptive immune responses. The DNA methylation profiles between CR and Non-CR groups differ significantly, and we speculate that the methylation status of 8 genes may have predictive value for treatment efficacy.