第一部分 单药免疫抑制剂在大动脉炎维持治疗中的疗效比较研究

背景与目的 大动脉炎（Takayasu arteritis, TAK）的复发率高，但关于TAK缓解期维持治疗最佳的药物选择尚不明确。本研究的目的是在真实世界条件下比较不同的传统免疫抑制剂在TAK缓解期作为维持治疗预防复发的有效性和安全性。

对象与方法 本研究为单中心回顾性队列研究，纳入符合TAK诊断标准并处于疾病缓解期、糖皮质激素剂量≤10mg/天泼尼松（或等效剂量）、联合使用单药免疫抑制剂的患者，以缓解期开始使用单药免疫抑制剂的时间作为基线时间，收集患者人口学资料、临床特征、用药情况及每次随访时的资料。结局定义为复发或稳定。比较不同单药免疫抑制剂治疗的有效性。采用Kaplan-Meier法绘制单因素生存曲线，Log-rank检验比较组间生存率差异，单因素Cox回归筛选可能影响复发的危险因素，多因素Cox回归校正混杂因素，计算风险比（HR）和95%置信区间（CI）。

结果 研究共纳入232例次符合研究标准的患者，其中207例次（89.2%）为女性，中位起病年龄为26.0（20.9-31.6）岁。霉酚酸酯组60例次（25.9%），硫唑嘌呤组75例次（32.3%），甲氨蝶呤组97例次（41.8%）。所有患者从基线到研究结局的中位随访时间为26.8（16.7-44.5）个月，共69例次（29.7%）在随访期间出现复发，其复发时间的中位数为19.1（11.1-26.4）个月，在中位随访时间时的累积复发概率为27.2%。3组间的重症复发率未发现显著差异（P=0.962）。以霉酚酸酯组作为参考组，Kaplan-Meier生存曲线显示3组间的复发率存在显著差异（Log-rank P=0.007）。在随访24个月时，霉酚酸酯组、硫唑嘌呤组和甲氨蝶呤组的估算累积复发率分别为35.9%，16.1%和17.8%。在随访48个月时，3组估算的累积复发率分别为57.3%，35.6%和34.7%。对混杂因素进行校正后，基线时血管受累严重程度是影响复发的独立危险因素（HR=1.048，95% CI 1.001-1.096，P=0.044）。在校正血管受累严重程度后，与霉酚酸酯组相比，硫唑嘌呤组的复发风险降低了54.9%（HR=0.451，95% CI 0.249-0.818，P=0.009），甲氨蝶呤组降低了51.7%（HR=0.483，95% CI 0.277-0.844，P=0.011）。随访过程中，有72例次尝试减停激素，其中9例次复发（12.5%），55例次最终达到无激素缓解，占所有患者的23.7%。有12例次停用单药免疫抑制剂，单用小剂量激素维持治疗，其中4例次复发（33.3%）。随访期间，甲氨蝶呤的主要不良反应为肝功能异常（7.2%），硫唑嘌呤为骨髓抑制（5.3%），霉酚酸酯为肝功能异常（1.7%）。

结论 相比霉酚酸酯，硫唑嘌呤和甲氨蝶呤在TAK缓解期维持治疗中预防复发的疗效更好。血管受累严重程度是影响TAK缓解期复发的独立危险因素。近1/4的患者在维持缓解期可以达到无激素缓解。3种药物在维持治疗期间未造成严重不良反应。

第二部分 大动脉炎患者血清IgG糖基化特征改变

背景与目的 大动脉炎（Takayasu arteritis, TAK）是一种自身免疫性炎症性疾病，其病因尚未完全阐明。凝集素芯片是一种新型的糖基化分析工具，可以快速、灵敏、高通量地获得蛋白总体的糖基化谱。本研究旨在描述TAK患者血清免疫球蛋白G（IgG）的糖基化改变。

材料与方法 发现队列共纳入164例TAK患者（包括82例活动期和82例稳定期患者），128例动脉粥样硬化患者（作为疾病对照）和100例健康对照。采用包被有56种凝集素的凝集素芯片检测血清IgG的糖基化水平。比较TAK、动脉粥样硬化患者和健康对照间的糖基化水平，寻找疾病特异性糖基化改变；比较活动期和稳定期TAK患者间的糖基化水平，寻找疾病状态相关糖基化改变。在凝集素印迹实验中对差异性糖基化改变进行进一步验证。采用受试者工作特征曲线（ROC）分析评估疾病特异性糖基化改变鉴别TAK和动脉粥样硬化的效能。

结果 凝集素芯片实验发现TAK患者血清IgG的N-乙酰半乳糖胺（GalNAc）水平显著高于疾病对照组和健康对照（P均＜0.01），而动脉粥样硬化患者和健康对照间GalNAc水平无显著差异；活动期TAK患者血清IgG甘露糖糖基化水平较稳定期患者明显减低（P＜0.01）。以上差异性糖基化改变在凝集素印迹实验中得到了验证。GalNAc水平在鉴别TAK和动脉粥样硬化方面显示出了良好的效能，ROC曲线下面积为0.749（P＜0.001），在最佳界值处的敏感性为71.7%，特异性为73.8%。

结论 TAK患者血清IgG存在疾病特异性的糖基化改变，该糖基化改变有望成为鉴别TAK和动脉粥样硬化的生物学标志物。异常的糖基化改变可能与IgG的促炎性相关，为体液免疫在TAK发病机制中的作用提供了新的猜想。

第三部分 单细胞转录组测序揭示初治大动脉炎外周血NK细胞CD160表达增高

背景与目的 大血管炎的发病是固有免疫和适应性免疫持续激活的共同结果，但目前有关固有免疫细胞在大动脉炎发病机制中的作用的研究还很有限。本部分研究旨在对初治大动脉炎患者外周血单个核细胞进行单细胞水平的转录组测序研究，初步探究大动脉炎发病早期免疫细胞的免疫学特征和功能改变。

材料与方法 采集5例初治大动脉炎患者和2例性别、年龄匹配的健康对照的外周血，分离出单个核细胞，利用10× Genomics单细胞测序平台进行3’单细胞转录组测序。利用生物信息学分析工具，对单细胞数据进行降维、聚类分析并可视化，绘制外周血单个核细胞免疫图谱。对初治大动脉炎及健康对照各细胞亚群的基因表达进行差异性分析，构建差异性基因表达谱。应用流式细胞术对筛选出的候选基因在初治大动脉炎、非初治活动期大动脉炎、稳定期大动脉炎患者和健康对照中进行验证。

结果 7个样本经过质控和过滤后总共有80791个细胞纳入分析，最终经过降维、聚类，分成4大类23群细胞。分别提取T/NK细胞群、B细胞群、单核/树突状细胞群的数据进行进一步分析。结果发现初治大动脉炎患者的调节性T细胞（Tregs）、1型经典树突状细胞（cDC1）细胞的比例有降低趋势，而干扰素反应性B细胞、浆细胞、CD56bright自然杀伤细胞、CD1c-CD141-树突状细胞和浆细胞样树突状细胞的比例有升高趋势。在CD56dim自然杀伤细胞中，初治大动脉炎患者的CD160表达明显高于健康对照，在流式细胞术中验证出了同样的结果。

结论 相比于健康对照，初治大动脉炎患者外周血的多种免疫细胞存在比例失衡，这可能是导致免疫调节失衡的原因。初治大动脉炎患者细胞毒性的CD56dim自然杀伤细胞比例减低，但其CD160表达明显升高，CD160的高表达可能导致干扰素-γ等促炎细胞因子分泌增强，参与大动脉炎发病。

Part I Comparison of the efficacy of 3 immunosuppressants monotherapy for maintenance treatment in Takayasu arteritis

Background and Objective Takayasu arteritis (TAK) presents high rates of relapse. However, the optimal therapeutic agents for the maintenance of disease remission remain unclear. This study aimed to investigate the efficacy and safety of 3 different conventional immunosuppressants, as maintenance therapy, in preventing relapse during the remission period under real-world conditions.

Material and Methods The present study was a single-center, retrospective cohort study. TAK patients who were in remission and were given glucocorticoids (≤10mg/day prednisone or equivalent) and immunosuppressant monotherapy were included. The beginning of taking immunosuppressant monotherapy during the remission phase was taken as the baseline time point. Demographic data, clinical characteristics, treatment regimen, and follow-up data were collected. Study outcomes were defined as relapse or no relapse. The efficacy of maintenance treatment was compared among mycophenolate mofetil (MMF), azathioprine (AZA) and methotrexate (MTX). Univariate survival curves were drawn by the Kaplan-Meier methods, and the statistical difference between survival curves was assessed by the Log-rank test. Univariate Cox regression analysis was used to screen the risk factors of TAK recurrence. Multivariate Cox proportional hazards regression analysis was used to adjust for confounders and calculate the hazard ratio (HR) of each factor with 95% confidence intervals (CI).

Results A total of 232 cases who met the inclusion criteria were enrolled in the study, 207 were females (89.2%) and the median (IQR) onset age was 26.0 (20.9-31.6) years. Sixty cases used MMF (25.9%), 75 used AZA (32.3%), and 97 used MTX (41.8%). The median (IQR) follow-up period from baseline to the study’s endpoint was 26.8 (16.7-44.5) months. A total of 69 cases experienced recurrence during the follow-up period and their median (IQR) time to recurrence was 19.1 (11.1-26.4) months. The cumulative recurrence rate at 26.8-month follow-up was 27.2%. No significant difference was observed in the major relapse rate among the 3 groups (P=0.962). Using MMF as the reference group, Kaplan-Meier survival curves revealed a significant difference among the 3 groups (Log-rank P=0.007). The estimated cumulative recurrence rate for MMF, AZA and MTX at 24-month follow-up was 35.9%, 16.1% and 17.8%, respectively, and 57.3%, 35.6% and 34.7%, respectively, at 48-month follow-up. After adjusting for confounders, the severity of vascular involvement was an independent risk factor for TAK relapse (HR=1.048, 95% CI 1.001-1.096, P=0.044). After adjusting for the severity of vascular involvement, AZA showed a 54.9% decreased risk of relapse (HR= 0.451, 95% CI 0.249-0.818, P=0.009), and MTX showed a 51.7% decreased risk of relapse (HR=0.483, 95% CI 0.277-0.844, P=0.011), compared with MMF. During the follow-up period, 72 cases attempted to taper off glucocorticoids. Of them, 9 cases relapsed (12.5%), and 55 cases achieved glucocorticoid-free remission (23.7% of all cases). Twelve cases withdrew non-glucocorticoid immunosuppressants and retain low-dose glucocorticoid monotherapy, and 4 cases of them relapsed (33.3%). During follow-up, adverse reactions were mainly recorded as an abnormal hepatic function for MTX (7.2%) and MMF (1.7%), and myelosuppression for AZA (5.3%).

Conclusions Compared with MMF, AZA and MTX showed significantly better relapse prevention as maintenance therapies while in the remission periods of TAK. The severity of vascular involvement was an independent risk factor for TAK relapse. Nearly a quarter of cases could achieve glucocorticoid-free remission during remission maintenance periods. No severe adverse drug reaction occurred with MMF, AZA and MTX during treatment.

Part II Altered glycosylation profiles of serum IgG

in Takayasu arteritis

Background and Objective Takayasu arteritis (TAK) is an autoimmune inflammatory disorder with an undefined etiology. The lectin microarray is a novel tool for glycan analysis that enables obtaining global glycosylation patterns in a rapid and highly sensitive way. This study aimed to characterize the glycosylation profiles of serum immunoglobulin G (IgG) in patients with TAK.

Material and Methods The discovery cohort included 164 patients with TAK (82 active and 82 inactive), 128 patients with atherosclerosis used as disease controls (DCs), and 100 healthy controls (HCs). Lectin microarrays containing 56 types of lectins were used to detect the glycan levels of serum IgG. Differentially altered glycosylation patterns between TAK and control groups as well as between TAK subgroups were identified and further validated by lectin blot. The classification performance of the TAK-specific glycosylation change was measured by receiver-operating characteristic (ROC) curve analysis.

Results Lectin microarray analysis revealed significantly increased N-Acetylgalactosamine (GalNAc) levels in the TAK group compared to the DC and HC groups (all P＜0.01). No significant difference was found between the DC and HC groups (P＞0.99). For TAK subgroups, significantly decreased mannosylation was observed in patients with active TAK compared to patients with inactive disease (P＜0.01). These differences were validated by lectin blot. In addition, GalNAc levels exhibited a considerable potential for discriminating patients with TAK from patients with atherosclerosis, with an area under the curve of 0.749 (P＜0.001), a sensitivity of 71.7%, and a specificity of 73.8%.

Conclusions Serum IgG in patients with TAK displayed disease-specific glycosylation alterations. Aberrant GalNAc glycosylation showed substantial value as a diagnostic biomarker. The potential proinflammatory properties of the abnormal glycans may provide new insights into the role of humoral immunity in the pathogenesis of TAK.

Part III Single-cell RNA sequencing revealed increased CD160 expression on circulating natural killer cells in untreated Takayasu arteritis

Background and Objective Sustained activation of the innate and adaptive immune responses cooperatively lead to the pathogenesis of Takayasu arteritis(TAK). However, the research regarding the role of innate immune cells in TAK pathogenesis is currently limited. The present study performed single-cell RNA sequencing using peripheral blood mononuclear cells (PBMC) of untreated patients with TAK and aimed to primarily explore the alterations of immunological profiles and function of immune cells in the early stages of TAK.

Material and Methods PBMC were collected from 5 untreated TAK patients and 2 gender- and age-matched healthy controls. 10× Chromium single-cell platform (10× Genomics) was used to perform single-cell RNA sequencing. Bioinformatics tools were used to conduct reduction analysis and clustering analysis and visualize single-cell RNA-seq data. The immunological profiles of immune cells in PBMC were described. Differential gene expression analysis was performed between untreated TAK and healthy controls for each cell cluster. Flow cytometry was used to validate the selected candidate gene in untreated TAK, treated-active TAK, treated-inactive TAK patients and healthy controls.

Results In total, 80791 cells from 7 samples were retained for downstream analysis after quality control and filtration. Dimensionality reduction analysis identified 23 major clusters of 4 main categories. Data of T/Natural killer (T/NK) cells, B cells, and Monocytes/Dendritic cells were extracted separately and further analyzed. The fraction of regulatory T cells (Tregs), type 1 classical dendritic cells (cDC1) tended to decrease, and that of interferon(IFN)-activated B cells, plasma cells, CD56bright NK cells, CD1cCD141- dendritic cells, and plasmacytoid dendritic cells (pDC) tended to increase in untreated TAK. CD160 expressions on CD56dim NK cells were significantly higher in untreated TAK than that in healthy controls, and this result was verified using flow cytometry.

Conclusions Compared with healthy controls, there are imbalances in the proportion of multiple peripheral immune cells in untreated TAK, which might be a possible reason for disturbed immune regulation. Although the fraction of cytotoxic CD56dim NK cells is decreased in PBMC of untreated TAK, the CD160 expression is remarkably increased. The overexpression of CD160 might lead to enhanced secretion of pro-inflammatory cytokines, such as IFN-γ, and thus participates in TAK pathogenesis.