第一部分第一章

目的：袖式肺叶切除术（Sleeve Lobectomy，SL）正逐步取代全肺切除术（Pneumonectomy，PN）成为部分特定的中心型原发性非小细胞肺癌（Non-Small Cell Lung Cancer，NSCLC）患者当下最优选的外科手术方式。本章节研究旨在分析可切除中心型NSCLC患者袖式肺叶切除术后不同亚组的长期临床结局，评估影响NSCLC-SL患者生存及复发的临床特征和预后因素，探讨新辅助治疗（Neoadjuvant Therapy，NT）对患者预后及围术期风险的影响，并对新辅助治疗的安全性及有效性进行初步探索。

方法：回顾性纳入国家癌症中心/中国医学科学院肿瘤医院胸外科2010年1月至2023年12月行支气管袖式肺叶切除+纵隔及肺门淋巴结清扫术的288例原发性中心型NSCLC患者。使用Kaplan-Meier法进行生存分析并绘制各亚组患者生存曲线，单多因素COX回归模型识别预后影响因素。根据是否接受新辅助治疗将患者分为85例接受新辅助治疗的SL（Neoadjuvant Therapy Sleeve Lobectomy，NT-SL）患者和203例未接受新辅助治疗的SL（Non-Neoadjuvant Therapy Sleeve Lobectomy，Non-NT-SL）患者，通过倾向评分匹配（Propensity Score Matching，PSM）以平衡组间基线差异，比较两组围术期指标及长期生存结局。进一步亚组分析评估不同新辅助治疗类型及不同病理反应程度的疗效差异。结果：生存分析表明，NT-SL患者较Non-NT-SL患者总生存期（Overall Survival，OS，Log-rank P=0.004）和无病生存期（Disease-Free Survival，DFS，Log-rank P=0.01）显著获益，且不增加围术期相关风险，而PSM后组间预后失去统计学差异。接受新辅助化免治疗的SL（Neoadjuvant Chemoimmunotherapy Sleeve Lobectomy，NCIT-SL）患者较接受新辅助化疗的SL（Neoadjuvant Chemotherapy Sleeve Lobectomy，NCT-SL）患者OS（Log-rank P=0.22）和DFS（Log-rank P=0.82）有获益趋势，且围术期风险相当，但获益未达到统计学差异。新辅助治疗后病理完全缓解（Complete Pathological Response，CPR）的SL患者OS（Log-rank P=0.17）和DFS（Log-rank P=0.19）较未病理完全缓解（Non-Complete Pathological Response，Non-CPR）的SL患者有获益趋势，病理完全缓解和主要病理缓解（Major Pathological Response，MPR）的SL患者OS（Log-rank P=0.1）较Non-CPR/MPR的SL患者亦有获益趋势，但预后差异均未达到统计学意义，而仅CPR+MPR组SL患者DFS（Log-rank P=0.037）较Non-CPR/MPR组SL患者有显著获益。不同亚组预后分析表明，高体重指数（Body Mass Index, BMI）的SL患者OS（Log-rank P=0.005）及DFS（Log-rank P=0.014）显著高于低BMI患者，肿瘤位于不同肺叶位置的患者OS及DFS也存在显著差异（Log-rank P<0.001）。此外，不同病理肿瘤大小的预后显著不同，肿瘤>3cm的患者的OS（Log-rank P=0.03）及DFS（Log-rank P=0.001）均显著低于肿瘤≤3cm的患者，而鳞癌（Squamous Cell Carcinoma，SCC）及其他病理类型的DFS均较腺癌（Adenocarcinoma，ADC）显著获益（Log-rank P=0.02）。在TNM分期方面，cT分期越高的患者DFS显著较差（Log-rank P<0.001），而OS无统计学差异（Log-rank P=0.07），其他临床分期亦无统计学差异。而不同pT、pN、pTNM患者预后差异显著不同（Log-rank P<0.05），均表现为分期越高预后越差。单多因素COX回归分析表明，BMI（P=0.012）和新辅助治疗（P=0.005）是SL患者OS的独立预后因素，而BMI（P=0.012）、临床T分期（P=0.031）、病理N分期（P=0.024）和新辅助治疗（P=0.012）则是SL患者DFS的独立预后因素。

结论：新辅助免疫治疗和/或化疗可以显著改善NSCLC-SL患者长期生存，且而不增加围术期风险。第9版TNM分期更能真实反映SL患者的预后，而N分期是预测接受袖式切除的NSCLC患者远期复发风险的重要参考，彻底的纵隔和肺门淋巴结清扫对于降低复发风险和改善长远预后尤为重要。尽管新辅助免疫治疗联合化疗显示出潜在优势，仍需通过多中心、大样本临床研究来进一步验证其疗效。

第一部分第二章 目的：本章节通过多中心大样本真实世界队列研究，评估NSCLC-SL患者围术期风险相关指标及长期生存结局。本研究拟基于第一章内容，进一步验证新辅助治疗在可切除中心型NSCLC袖式肺叶切除术患者中的安全性和有效性，明确影响新辅助治疗后NSCLC-SL患者生存及复发的临床特征和预后因素。方法：回顾性纳入2010年1月至2023年12月国内6家大型区域医疗中心接受袖式肺叶切除+纵隔及肺门淋巴结清扫术的462名原发性中心型NSCLC患者，其中153例接受新辅助治疗（NT-SL组），309例未接受新辅助治疗（Non-NT-SL组）。按治疗类型（NCT-SL组 vs. NCIT-SL组）、手术时期（2010-2020组 vs. 2021-2023组）及病理反应（CPR组 vs. Non-CPR组/CPR+MPR组 vs. Non-CPR/MPR组）分层，进一步对NT-SL组进行亚组分析。采用PSM平衡混杂因素，根据不同匹配变量建立平衡队列，比较组间围术期指标及生存结局，COX回归模型识别预后影响因素。结果：在总队列中，NT-SL患者比Non-NT-SL患者拥有更高的临床T分期（P=0.002），N分期（P<0.001）及TNM分期（P<0.001）。按是否接受新辅助治疗进行PSM后的平衡队列消除了主要差异，Kaplan-Meier分析表明，NT-SL患者相比Non-NT-SL患者拥有更好的OS（Log-rank P=0.003）和DFS（Log-rank P=0.003）。PSM后队列的COX多因素回归分析表明，性别（HR=0.469, 95%CI=0.246-0.894，P=0.022）、新辅助治疗（HR=0.486，95%CI=0.301-0.786，P=0.003）、以及临床T分期（P=0.016）是SL患者OS的独立预后因素，而BMI（HR=0.518，95%CI=0.326-0.824，P=0.005）、新辅助治疗（HR=0.481，95%CI=0.286-0.809，P=0.006）、临床T分期（P=0.020）是SL患者DFS的独立预后因素。NT-SL患者按新辅助治疗类型进行PSM后的平衡队列中，Kaplan-Meier分析表明，NCIT-SL患者比NCT-SL患者的OS（Log-rank P=0.03）显著获益，而DFS（Log-rank P=0.68）未见明显差异。按新辅助治疗类型PSM后群体的COX分析表明，新辅助治疗类型（HR=0.271，95%CI=0.076-0.961，P=0.043）、ypT分期（HR=5.038，95%CI=1.61-15.767，P=0.005）、ypTNM分期（HR=4.392，95%CI=1.588-12.143，P=0.004）是接受新辅助治疗SL患者OS的影响因素，而新辅助治疗类型（HR=0.254，95%CI=0.066-0.972，P=0.045）是其独立预后因素。按手术时期进行PSM后的平衡队列中，2021-2023组相比2010-2020组的手术患者表现出更高的NCIT比例（P<0.001）、更小的肿瘤大小（P=0.001）、更低的术后病理分期（P=0.010）及更好的病理学反应（P=0.010），腔镜及中转率却更高（P=0.010）。新辅助治疗后不同病理反应程度的SL患者总生存期（Log-rank P=0.025)存在显著差异，CPR患者预后最佳，而DFS（Log-rank P=0.32)未达到统计学差异。CPR组SL患者总生存期（Log-rank P=0.016/0.002）在PSM前后均较Non-CPR组SL患者显著获益，而无病生存期（Log-rank P=0.1/0.0096）PSM前后预后差异均未达到统计学意义。CPR+MPR组SL患者总生存期（Log-rank P=0.007）较Non-CPR组SL患者显著获益，而无病生存期（Log-rank P=0.078）有获益趋势，未达到统计学差异。而PSM后OS及DFS均未达到统计学差异（P>0.05）。新辅助治疗SL患者不同亚组预后分析表明，肿瘤>3cm的新辅助SL患者OS（Log-rank P=0.008）显著低于肿瘤≤3cm的患者。在TNM分期方面，不同cT、cN、cTNM分期患者预后均未达统计学差异（Log-rank P>0.05）。不同pT、pN 8th、pTNM 8th/9th分期的新辅助治疗SL患者OS（Log-rank P<0.05）显著不同，DFS（Log-rank P>0.05）均无统计学差异，而不同pN 9th分期患者OS（Log-rank P=0.012）和DFS（Log-rank P=0.004）均存在差异，表现为多站纵隔淋巴结转移pN2b期患者预后最差。接受新辅助治疗的153例SL患者群体的单多因素COX回归分析表明，病理TNM分期（HR=2.512，95%CI=1.045-6.038，P=0.040）是SL患者DFS的独立预后因素。结论：本章节研究首次验证了新辅助治疗在可切除中心型NSCLC袖式肺叶切除患者中的安全性及有效性。新辅助免疫治疗联合化疗显著改善袖式肺叶切除术患者的长期生存，且不增加围术期风险，推荐符合新辅助治疗指征的NSCLC-SL患者优先选择NCIT，并以病理TNM分期指导术后管理。本章节为目前全球最大样本量的多中心SL患者预后的真实世界队列研究，为优化NSCLC-SL患者新辅助治疗策略提供了高级别证据支持。

第二部分

目的：本研究旨在探究滤泡辅助T细胞（T follicular helper cell，Tfh）在非小细胞肺癌免疫治疗中的作用机制，聚焦CD53基因对Tfh分化的调控及其对肿瘤微环境（Tumor Microenvironment，TME）免疫应答的影响，探索NSCLC新辅助免疫治疗疗效的相关生物标志物，以期为优化免疫治疗策略提供理论依据和潜在靶点。

方法：本部分研究纳入2024年1月至6月于国家癌症中心/中国医学科学院肿瘤医院行新辅助免疫治疗后38例NSCLC患者（CPR+MPR组23例，Non-CPR/MPR组15例）肿瘤组织样本，通过Bulk RNA测序分析TME的基因表达差异。同时整合GEO（Gene Expression Omnibus）数据库中1072例非免疫治疗样本及39例免疫治疗后NSCLC患者样本（单细胞RNA测序15例、Bulk RNA测序24例）进行验证。通过生存分析探究病理反应与预后的关联，采用肿瘤免疫功能障碍和排斥（Tumor Immune Dysfunction and Exclusion，TIDE）评分系统评估肿瘤免疫逃逸机制。通过limma包筛选差异基因，并进行GO/KEGG功能富集分析、基因集富集分析（GSVA/ssGSEA/GSEA）解析通路活性。单细胞测序数据经质控、批次校正后，利用UMAP分群和Monocle2聚类及拟时序分析，解析Tfh分化轨迹及关键调控基因的动态表达。

结果：CPR+MPR组患者的总生存期和无复发生存期显著优于Non-CPR/MPR组。差异基因及GO/KEGG功能富集分析显示，CPR+MPR组中免疫激活相关通路显著上调，而细胞周期相关通路下调，提示病理反应好的患者TME中免疫应答增强与肿瘤增殖抑制。差异分析显示CPR+MPR组相比Non-CPR/MPR组Tfh细胞浸润明显上升（P<0.01），且Tfh细胞与活化CD8+T细胞（R=0.82）及效应记忆CD8+T细胞（R=0.90）呈强正相关（P<0.001），提示Tfh细胞高浸润水平可能与NSCLC免疫治疗良好的病理反应密切相关。Tfh细胞高浸润组的T细胞排斥（Exclusion）评分及MDSC/M2细胞浸润水平显著降低，尽管T细胞功能障碍（Dysfunction）评分升高，但Tfh细胞高浸润组的CD8+T细胞毒性评分和免疫水平显著升高（P<0.05），考虑这可能与T细胞募集数量增加相关，仍可表明Tfh高浸润组肿瘤微环境中存在更为活跃的抗肿瘤免疫反应。Tfh细胞高浸润组KRAS_SIGNALING_UP信号通路，IL6_JAK_STAT3信号通路，P53_PATHWAY信号通路以及EMT、肿瘤增殖率、缺氧及糖酵解活性等生物学行为被明显抑制，并且泛素介导的蛋白水解、叶酸介导的单碳代谢、半乳糖代谢、嘧啶代谢等促癌代谢行为也被抑制，从而进一步遏制肿瘤免疫逃逸。验证队列分析鉴定出36个与Tfh细胞浸润高度相关的基因（R>0.7，P<0.05）。单细胞分析显示Tfh细胞和CD4+T细胞分化发育密切相关，有共同的祖细胞。而CD53的差异表达参与了两种细胞分化和发育，其随着Tfh细胞发育和成熟逐渐上调而随总CD4+T细胞群发育和成熟逐渐下调。表明CD53可能通过调控幼稚CD4+T细胞向Tfh分化从而重塑TME。

结论：Tfh细胞通过打破TME中T细胞浸润屏障并募集更多的CD8+T细胞毒进入肿瘤微环境，显著改善NSCLC患者的免疫治疗病理反应。CD53高表达可能对驱动幼稚CD4+T细胞向Tfh细胞分化进而重塑肿瘤免疫微环境至关重要。本研究为Tfh细胞作为预测NSCLC免疫治疗疗效的生物标志物提供了理论依据，也为靶向CD53-Tfh轴的联合免疫治疗策略提供了关键分子机制和潜在干预靶点。

Part I Chapter 1.

Objectives: Sleeve lobectomy(SL) has gradually replaced pneumonectomy(PN) as the currently optimal surgical option for certain specific patients with central primary non-small cell lung cancer (NSCLC). This chapter aims to analyze the long-term clinical outcomes of different subgroups of resectable central NSCLC patients after sleeve lobectomy, evaluate the clinical characteristics and prognostic factors affecting the survival and recurrence of NSCLC-SL patients, explore the impact of neoadjuvant therapy(NT) on patient prognosis and perioperative risks, and conduct a preliminary exploration of the safety and efficacy of neoadjuvant therapy.

Methods: A retrospective study included 288 patients with primary central NSCLC who underwent bronchial sleeve lobectomy plus mediastinal and hilar lymph node dissection in the Department of Thoracic Surgery, National Cancer Center/Cancer Hospital Chinese Academy of Medical Sciences, from January 2010 to December 2023. Kaplan-Meier method was used for survival analysis and to plot the survival curves of patients in each subgroup. Univariate and multivariate COX regression models were used to identify prognostic factors. Patients were divided into 85 NT-SL patients who received neoadjuvant therapy and 203 Non-NT-SL patients who did not receive neoadjuvant therapy. Propensity score matching(PSM) was used to balance baseline differences and compare perioperative indicators and long-term survival outcomes between the two groups. Further subgroup analysis was conducted to evaluate the differences in efficacy of different types of neoadjuvant therapy and different degrees of pathological response.

Results: Survival analysis showed that NT-SL patients had significantly better overall survival(OS, Log-rank P=0.004) and disease-free survival(DFS, Log-rank P=0.01) than Non-NT-SL patients, and did not increase perioperative-related risks. However, after PSM, the prognosis difference between the groups lost statistical significance. Patients with SL who received neoadjuvant chemoimmunotherapy(NCIT-SL) tended to have better OS(Log-rank P=0.22) and DFS(Log-rank P=0.82) than those who received neoadjuvant chemotherapy(NCT-SL), with equivalent perioperative risks, but the benefits did not reach statistical significance. SL patients who achieved a complete pathological response(CPR) after neoadjuvant therapy had a trend towards better OS(Log-rank P=0.17) and DFS(Log-rank P=0.19) than those who did not achieve CPR(Non-CPR). SL patients with both complete pathological response and major pathological response(MPR) also had a trend towards better OS(Log-rank P=0.1) than those with Non-CPR/MPR, but the prognosis differences did not reach statistical significance. Notably, only in the CPR + MPR group, SL patients had a significant benefit in DFS(Log-rank P=0.037) compared to those in the Non-CPR/MPR group.

Prognostic analysis of different subgroups showed that SL patients with a high body mass index(BMI) had significantly better OS(Log-rank P=0.005) and DFS(Log-rank P=0.014) than those with a low BMI. There were also significant differences in OS and DFS among patients with tumors located in different lobes(Log-rank P<0.001). In addition, the prognosis was significantly different for different pathological tumor sizes. Patients with tumors larger than 3cm had significantly worse OS(Log-rank P=0.03) and DFS(Log-rank P=0.001) than those with tumors ≤3cm. The DFS of squamous cell carcinoma(SCC) and other rare pathological types was better than that of adenocarcinoma(ADC, Log-rank P=0.02). In terms of TNM staging, patients with higher cT staging had significantly worse DFS(Log-rank P<0.001), while there was no statistical difference in OS(Log-rank P=0.07). There were also no statistical differences in other clinical stages. However, there were significant differences in prognosis among patients with different pT, pN, and pTNM(Log-rank P<0.05), all showing that the higher the stage, the worse the prognosis. Univariate and multivariate COX regression analysis showed that BMI(P=0.012) and neoadjuvant therapy(P=0.005) were independent prognostic factors for OS in SL patients, while BMI(P=0.012), clinical T staging(P=0.031), pathological N staging(P=0.024), and neoadjuvant therapy(P=0.012) were independent prognostic factors for DFS in SL patients.

Conclusions: Neoadjuvant immunotherapy and/or chemotherapy can significantly improve the long-term survival of NSCLC-SL patients without increasing perioperative risks. The 9th edition of TNM staging is more reflective of the prognosis of SL patients. N staging is an important reference for predicting the long-term recurrence risk of NSCLC patients undergoing sleeve resection. Thorough mediastinal and hilar lymph node dissection is particularly important for reducing the risk of recurrence and improving long-term prognosis. Although neoadjuvant immunotherapy combined with chemotherapy shows potential advantages, further validation of its efficacy is still needed through multicenter large-sample studies.

Part I Chapter 2.

Objectives: This chapter aims to evaluate the perioperative risk-related indicators and long-term survival outcomes of NSCLC-SL patients through a multicenter large sample real-world cohort study. It is intended to further validate the safety and efficacy of neoadjuvant therapy in resectable central NSCLC patients undergoing sleeve lobectomy based on Chapter 1, and to clarify the clinical characteristics and prognostic factors affecting the survival and recurrence of NSCLC-SL patients after neoadjuvant therapy.

Methods: A retrospective study included 462 patients with primary central NSCLC who underwent sleeve lobectomy plus mediastinal and hilar lymph node dissection in 6 large regional medical centers in China from January 2010 to December 2023. Among them, 153 SL patients received neoadjuvant therapy(NT-SL group), and 309 SL patients did not receive neoadjuvant therapy(Non-NT-SL group). Stratified by treatment type(NCT-SL group vs. NCIT-SL group), surgical period(2010-2020 group vs. 2021-2023 group), and pathological response(CPR group vs. Non-CPR group/CPR+MPR group vs. Non-CPR/MPR group), further subgroup analysis was conducted on the NT-SL group. PSM was used to balance confounding factors, and balanced cohorts were established according to different matching variables to compare perioperative indicators and survival outcomes between the two groups. COX regression model was used to identify prognostic factors.

Results: In the total cohort, NT-SL patients had higher clinical T staging(P=0.002), N staging(P<0.001), and TNM staging(P<0.001) than Non-NT-SL patients. The balanced cohort after PSM based on whether neoadjuvant therapy was received eliminated the main differences. Kaplan-Meier analysis showed that NT-SL patients had better OS(Log-rank P=0.003) and DFS(Log-rank P=0.003) than Non-NT-SL patients. The multivariate COX regression analysis of the PSM-cohort showed that gender(Hazard Ratio[HR]=0.469, 95% Confidence Interval[CI]=0.246-0.894, P=0.022), neoadjuvant therapy(HR=0.486, 95%CI=0.301-0.786, P=0.003), and clinical T staging(P=0.016) were independent prognostic factors for OS in SL patients, while BMI(HR=0.518, 95%CI=0.326-0.824, P=0.005), neoadjuvant therapy(HR=0.481, 95%CI=0.286-0.809, P=0.006), and clinical T staging(P=0.020) were independent prognostic factors for DFS in SL patients. In the balanced cohort of NT-SL patients after PSM based on neoadjuvant therapy type, Kaplan-Meier analysis showed that NCIT-SL patients had significantly better OS(Log-rank P=0.03) than NCT-SL patients, while there was no significant difference in DFS(Log-rank P=0.68). The COX analysis of the PSM-cohort based on neoadjuvant therapy type showed that neoadjuvant therapy type(HR=0.271, 95%CI=0.076-0.961, P=0.043), ypT staging(HR=5.038, 95%CI=1.61-15.767, P=0.005), and ypTNM staging(HR=4.392, 95%CI=1.588-12.143, P=0.004) were factors affecting OS in SL patients receiving neoadjuvant therapy, and neoadjuvant therapy type(HR=0.254, 95%CI=0.066-0.972, P=0.045) was an independent prognostic factor. In the balanced cohort after PSM based on surgical period, patients who underwent surgery in the 2021-2023 group had a higher proportion of NCIT(P<0.001), smaller tumor size(P=0.001), lower postoperative pathological staging(P=0.010), and better pathological response(P=0.010) than those in the 2010-2020 group, but the conversion rate and VATS rate was higher(P=0.010). There was a significant difference in overall survival(Log-rank P=0.025) among SL patients with different degrees of pathological response after neoadjuvant therapy, with the best prognosis in CPR patients, while DFS(Log-rank P=0.32) did not reach statistical significance. In the CPR group, the overall survival of SL patients(Log-rank P=0.016/0.002) was significantly better than that of the Non-CPR group before and after PSM, while disease-free survival(Log-rank P=0.1/0.0096) did not reach statistical significance in prognosis before and after PSM. The overall survival of SL patients in the CPR + MPR group(Log-rank P=0.007) was significantly better than that of the Non-CPR group, while disease-free survival(Log-rank P=0.078) had a trend of benefit but did not reach statistical significance. After PSM, neither OS nor DFS reached statistical significance(P>0.05). Prognostic analysis of different subgroups of SL patients receiving neoadjuvant therapy showed that the OS of patients with tumors larger than 3cm(Log-rank P=0.008) was significantly lower than that of patients with tumors ≤3cm. In terms of TNM staging, there was no significant difference in prognosis among patients with different cT, cN, and cTNM stages(Log-rank P>0.05). The OS of SL patients receiving neoadjuvant therapy with different pT, pN 8th, and pTNM 8th/9th stages(Log-rank P<0.05) was significantly different, while DFS(Log-rank P>0.05) had no significant difference. The OS(Log-rank P=0.012) and DFS(Log-rank P=0.004) of patients with different pN 9th stages were different, with the worst prognosis in patients with multi-station mediastinal lymph node metastasis in pN2b stage. The univariate and multivariate COX regression analysis of the 153 SL patients receiving neoadjuvant therapy showed that pathological TNM staging(HR=2.512, 95%CI=1.045-6.038, P=0.040) was an independent prognostic factor for DFS in SL patients. Conclusions: This chapter is the first to validate the safety and efficacy of neoadjuvant therapy in resectable central NSCLC patients undergoing sleeve lobectomy. Neoadjuvant immunotherapy combined with chemotherapy significantly improves the long-term survival of patients undergoing sleeve lobectomy without increasing perioperative risks. It is recommended that NSCLC-SL patients who meet the indications for neoadjuvant therapy prioritize NCIT and guide postoperative management with pathological TNM staging. This chapter is the largest-sample multicenter real-world cohort study on the prognosis of SL patients to date, providing high-level evidence support for optimizing neoadjuvant therapy strategies. Keywords: Sleeve lobectomy, Neoadjuvant chemoimmunotherapy, Long-term prognosis, Perioperative risks, Real-world study. Part II. Objectives: This study aims to explore the mechanism of T follicular helper cells(Tfh) in the immunotherapy of non-small cell lung cancer(NSCLC), focusing on the regulation of Tfh differentiation by the CD53 gene and its impact on the immune response in the tumor microenvironment(TME). It also seeks to identify potential biomarkers related to the efficacy of neoadjuvant immunotherapy for NSCLC, in order to provide a theoretical basis and potential targets for optimizing immunotherapy strategies. Methods: This part of the study included tumor tissue samples from 38 NSCLC patients who received neoadjuvant immunotherapy at the National Cancer Center/Cancer Hospital Chinese Academy of Medical Sciences, from January to June 2024(23 cases in the CPR+MPR group and 15 cases in the Non-CPR/MPR group). Bulk RNA sequencing was used to analyze the differences in gene expression in the TME. Meanwhile, 1072 non-immunotherapy samples and 39 immunotherapy-treated NSCLC patient samples(15 cases of single-cell RNA sequencing and 24 cases of Bulk RNA sequencing) from the GEO database were integrated for validation. Survival analysis was conducted to explore the association between pathological response and prognosis, and the TIDE scoring system was used to assess tumor immune escape mechanisms. The limma package was used to select differentially expressed genes, and GO/KEGG functional enrichment analysis, gene set enrichment analysis(GSVA/ssGSEA/GSEA) were performed to parse pathway activity. After quality control and batch correction, single-cell sequencing data were clustered using UMAP and Monocle2, and pseudo-time sequence analysis was carried out to parse the differentiation trajectory of Tfh cells and the dynamic expression of key regulatory genes. Results: The overall survival(OS) and recurrence-free survival(DFS) of patients in the CPR+MPR group were significantly better than those in the Non-CPR/MPR group. The analysis of differentially expressed genes and GO/KEGG functional enrichment analysis showed that immune-activation-related pathways were significantly up-regulated in the CPR+MPR group, while cell-cycle-related pathways were down-regulated, suggesting that enhanced immune response and inhibited tumor proliferation in the TME of patients with good pathological response. The difference analysis showed that the infiltration of Tfh cells in the CPR+MPR group was significantly higher than that in the Non-CPR/MPR group(P<0.01), and Tfh cells were strongly positively correlated with activated CD8+T cells(R=0.82) and effector memory CD8+T cells(R=0.90, P<0.001), indicating that high infiltration of Tfh cells may be closely related to good pathological response in NSCLC immunotherapy. In the group with high infiltration of Tfh cells, the T cell exclusion score and the infiltration level of MDSC/M2 cells were significantly reduced. Although the infiltration level of CAF and the T cell dysfunction score were increased, the latter may be related to the increased number of recruited T cells, and still indicated that high-level infiltration of Tfh cells can break through the barrier restricting T cell entry into the TME. The cytotoxicity score of CD8+T cells and the immune level were significantly increased in the group with high infiltration of Tfh cells(P<0.05), indicating that Tfh may enhance the function of CD8+T cells by secreting cytokines such as IL-21, and activated the tumor immune response. The KRAS_SIGNALING_UP pathway, IL6_JAK_STAT3 pathway, P53_PATHWAY, as well as EMT, tumor proliferation rate, hypoxia and glycolysis activity were significantly inhibited in the group with high infiltration of Tfh cells. Moreover, the carcinogenic metabolic behaviors such as ubiquitin mediated proteolysis, one carbon pool by folate, galactose metabolism, and pyrimidine metabolism were also inhibited, thus further curbing tumor immune escape. Cross-cohort analysis identified 36 genes highly correlated with Tfh cell infiltration(R>0.7, P<0.05). Single-cell pseudo-time sequence analysis showed that ALOX5AP, HLA-DPB1 and HLA-DRA were rapidly up-regulated in the late stage of Tfh cell development and maturation, while CD53 was gradually up-regulated with the development and maturation of Tfh cells. CD53 was gradually down-regulated with the development and maturation of total CD4+T cell population, while ALOX5AP, HLA-DPB1 and HLA-DRA had no obvious pattern. CD53 may reshape the TME by regulating the differentiation of naive CD4+T cells into Tfh cells.

Conclusions: Tfh cells significantly improve the pathological response and survival prognosis of NSCLC patients to immunotherapy by breaking through the T cell infiltration barrier in the TME and enhancing the cytotoxicity of CD8+T cells. CD53 may be a key regulatory gene inducing Tfh differentiation, and reshape the immune microenvironment by driving the differentiation of CD4+T cells into Tfh cells. This study provides a theoretical basis for Tfh cells as a biomarker for predicting the efficacy of NSCLC immunotherapy, and also provides key molecular mechanisms and potential intervention targets for targeted CD53-Tfh axis combined immunotherapy strategies.