第一部分：

研究背景与目的：T-大颗粒淋巴细胞白血病（T-LGLL）是一类罕见的慢性淋巴增殖性疾病，多呈惰性病程，以细胞毒性T淋巴细胞克隆扩增为特征，骨髓为主要受累器官，表现为血细胞减低，常伴发自身免疫性症状。免疫抑制治疗有效，但优势克隆持续存在。T-LGLL致病机制未完全阐明，本研究中我们拟通过单细胞转录组及免疫组库测序分析鉴定患者治疗前后免疫细胞的基因表达及T细胞克隆型。

方法：收集5例T-LGLL患者免疫抑制治疗前后及3例健康对照外周血，对表面蛋白进行特异性标记，使用10x genomics平台进行单细胞转录组联合配对的5’端V(D)J免疫组库建库，采用Illumina测序。使用Cell Ranger 6.1.0进行barcode赋值、比对、分子指数及T细胞受体序列组装。对免疫细胞进行聚类分群，解析各群细胞转录组特征，鉴定TCR克隆类型及克隆大小，分析差异基因并进行通路富集。

研究结果：总计测得67237个免疫细胞，22137个具有完整TCR α链与β链的T细胞。通过对转录组及免疫组库特征分析，发现：

（1）患者的抗原提呈细胞（APC）功能增强：1）单核细胞与树突状细胞均富集到抗原合成与呈递通路，而且与固有免疫相关的信号通路也上调；2）B淋巴细胞抗原结合能力增强，对T细胞活化、分化的调控通路上调。

（2）疾病状态自然杀伤（NK）细胞毒性相关基因表达下降，细胞溶解、杀伤通路下调，但对T细胞受体信号通路、T细胞增殖、T细胞活化及分化的调控作用增强。

（3）患者治疗前、无缓解状态免疫细胞构成类似，CD8 T细胞比例显著升高，该群细胞转录组与NK细胞转录组具有明显相似性。

（4）患者中处于G2/M期的效应CD8 T细胞（CD8_TE）比例明显高于健康对照；疾病状态的CD8_TE细胞中毒性相关基因、耗竭相关基因均上调表达，富集到细胞杀伤、干扰素γ信号通路及细胞因子分泌通路，治疗缓解后该群细胞增殖及毒性功能均下降。

（5）疾病状态CD8_TE细胞群克隆多样性显著减低，具有个体特异性的优势克隆扩增。而且克隆性CD8_TE细胞毒性功能更强，上调富集了T细胞活化、细胞因子分泌通路。治疗后优势克隆占比减低，缓解状态下优势克隆仍持续存在，但毒性功能下降。

（6）患者治疗前Treg比例低于健康对照，Treg细胞群富集凋亡相关通路上调，治疗缓解后Treg比例升高，代谢相关通路上调，凋亡通路下调。

研究结论：T-LGLL中存在免疫稳态失衡，APC的抗原呈递作用增强，对T细胞的活化、分化、增殖的调控能力增强；NK细胞毒性杀伤功能下降，但对T细胞的调控能力增强；CD8_TE细胞的TCR多样性减低，存在个体特异性的优势克隆，具有更强的细胞毒性与增殖能力；Treg细胞数目下调，功能失调。免疫抑制治疗能够部分清除致病大颗粒T淋巴细胞，使其毒性、增殖能力减低，丰富克隆多样性，恢复Treg功能、上调数目，改善免疫内环境。

第二部分：

研究背景与目的：原发获得性再生障碍性贫血（AA）是一类异常活化T细胞介导的骨髓衰竭性疾病，亚洲国家发病率相对高（0.6/百万/年）。根据疾病严重程度，分为重型再障（SAA）与非重型再障（NSAA）。NSAA发病率4倍于SAA，具有群体基数广、病情异质性大、治疗不统一、较高风险进展为SAA的特点。环孢素是治疗NSAA的首选药物，但总体反应率仅50%，而且缺乏疗效预测指标。为了更好地揭示NSAA免疫特点、细化分群、探索影响环孢素疗效的因素，我们设计了环孢素治疗NSAA的前瞻性队列，采用质谱流式技术分析骨髓T细胞免疫特点，以期为临床诊疗提供新思路。

方法：基于AA诊疗指南及文献回顾，设置纳排标准，入组AA患者，收集治疗前临床数据。均予环孢素3-5mg/kg/d治疗，满6月后评价疗效，分析临床特征。收集30例AA患者与4例健康对照骨髓样本，采用质谱流式检测技术分析骨髓T细胞免疫特征。收集7例患者治疗前后骨髓样本，采用质谱流式检测技术分析环孢素治疗前后T细胞免疫特征变化。

结果：我们建立了纳入92例患者的初治NSAA临床队列，细化了输血依赖型与非输血依赖型再障分类标准。79例患者接受环孢素治疗满6月，33例（41.8%）患者治疗后达到缓解，但通过治疗前血常规、生化等临床指标不足以预测环孢素治疗疗效。通过质谱流式检测技术，我们发现AA患者与健康人骨髓T细胞存在明显的免疫特征差异，AA患者中CD8⁺ T细胞亚群中细胞毒性T细胞相关蛋白-4 （CTLA-4）、初始T细胞中鞘氨醇-1-磷酸化受体1（S1P1）以及CD4⁺ T细胞亚群中白介素-9（IL-9）的表达均高于健康人。通过对环孢素治疗有效组与无效组T细胞免疫特征分析，我们发现相比治疗无效组，有效组治疗前CD8⁺ T细胞CTLA-4表达水平更低（16.59±1.83% vs 28.67±4.22%，p＜0.05），而且有效组治疗后CD8⁺ T细胞表面CTLA-4水平明显升高(33.94±6.38 % vs 11.28± 2.73 %, p < 0.05)。治疗有效组治疗前初始CD4细胞群中S1P₁（21.86±2.67% vs 32.18±3.46%，p＜0.05）、IL-9（24.52±2.97% vs 40.42±4.80%，p＜0.05）表达水平均低于治疗无效组。

结论：相比于健康对照，患者T细胞处于免疫功能失调的状态。环孢素有效组与无效组的T细胞中CTLA-4、S1P₁以及IL-9表达存在差异，而且有效组中CTLA-4表达在治疗后得到恢复。提示T细胞免疫检查点分子、T细胞迁移及分化标志有望用于预测免疫抑制治疗疗效，也为环孢素无效的患者提供了新的治疗方向。

Part 1:

Background and Objectives: T-cell large granular lymphocytic leukemia (T-LGLL) is a rare lymphoproliferative disorder, characterized by monoclonal T-cell large granular lymphocytosis, often clinically manifest as bone marrow failure and accompany with autoimmune symptoms. Immunosuppressive therapies are effective, but the dominant clone may persist even in responders. The pathogenesis of T-LGLL has not been fully elucidated. In this study, single-cell RNA sequencing (sc-RNA seq) and V(D)J profiling were performed to discern clonotypes and gene expression patterns of T lymphocytes from T-LGLL patients who were sampled before and after treatment.

Methods: Peripheral blood samples were collected from five T-LGLL patients prior and post immunotherapy treatment. Surface proteins were specifically labeled for epitopes sequencing, Single-cell RNA seq was performed on the 10x Genomics Chromium Single Cell V(D)J + 5' Gene Expression platform, and sequencing obtained on the Illumina Platform. Barcode assignment, alignment, unique molecular index counting and T cell receptor sequence assembly were performed using Cell Ranger 6.1.0. Immune cells were clustered and annotated, then transcriptome characteristics of each group of cells were analyzed, T cell receptor (TCR) clonetype and clonesize were identified. Furthermore, differential expression genes were identified and pathway enrichment was conducted.

Results: A total of 67,237 immune cells, including 22137 of T cells with intact TCRα and β chains were detected. By analyzing the characteristics of transcriptome and immune library, it was found that:

(1) The function of antigen presenting cells (APC) in patients was enhanced: 1）Antigen process and presentation pathway was enriched in monocytes and dendritic cells, the signal pathways related to innate immunity were also up-regulated. 2) For B lymphocyte, both the binding ability to antigen and the regulatory of T cell activation and differentiation were enhanced. 

(2) During disease condition, the expression of cytotoxicity related genes in NK cluster was decreased, and the pathways related to cytolysis and cytotoxicity were down-regulated, but the regulation of T cell receptor signaling pathway, T cell proliferation, activation and differentiation was enhanced. 

(3) The immune cell composition of patients before treatment and in non-response condition was similar, with increased proportion of CD8 T cell population. There was a more significant similarity on transriptome between CD8 T cells and NK cells.

(4) The proportion of effector CD8 T cells (CD8_TE) in G2/M phase was significantly higher in patients than in healthy individuals. Moreover, in patients prior treatment, cytotoxic genes of CD8 TE cluster was increased, exhaustion related genes were also upregulated. Cell killing, interferon γ signaling pathway and cytokine production were enhanced in CD8 TE from patients. But cell proliferation and toxicity of CD8 TE cluster in responders were decreased.

(5) The clonal diversity of CD8 TE cells was significantly reduced and the dominant clones were amplified. The dominant clones were individually specific and drifted with disease status. Increased cytotoxicity, up-regulated T cell activation and cytokine secretion were observed in monoclonal T cells. The proportion of dominant clones decreased after treatment, but the dominant clones still existed in remission state, with a decreased toxic function.

(6) In addition, for patient prior treatment, the proportion of Treg was decreased significantly and apoptosis pathways was enriched. After response, the proportion of Treg was restored, metabolism-related pathway was up-regulated and apoptosis-related pathways were down-regulated.

Conclusion: The immune homeostasis was unbalanced in T-LGLL. APCs demonstrated with increased antigen presentation effect and enhanced regulation on T cell activation, differentiation and proliferation. Stronger cytotoxicity and proliferation ability were observed in CD8 TE cluster. They also showed decreased diversity of TCR, and dominant clones was observed in individuals. NK cells was confirmed with less toxic killing function, but enhanced regulation ability to T cells. Decreased number and impaired function of Tregs may contribute pathogenesis of T-LGLL. IST can partially remove the pathogenic LGL cells, reduce their toxicity and proliferation ability, and enrich the clone diversity. The function of Treg was restored after IST, and the number of Treg was up-regulated.

Part 2:

Background and Objectives: Primary acquired aplastic anemia (AA) is a kind of bone marrow failure syndrome mediated by auto-activated T cells. The incidence of AA is relatively high in Asian countries (0.6/ million/year). According to the severity of the disease, it can be divided into severe aplastic anemia (SAA) and non-severe aplastic anemia (NSAA). NSAA is characterized by high-proportion (the incidence is four times of SAA), heterogeneous condition and high risk of progression to SAA. Cyclosporine is often used to treat with NSAA, but the overall response rate to cyclosporine is only 50%. Furthermore, it’s difficult to identify who will be responsive to cyclosporine. In order to better reveal the immune characteristics of NSAA and refine the subgroup, explore the factors affecting cyclosporine response and provide new methods for diagnosis and treatment. We designed a prospective cohort of NSAA, and analyzed the characteristics of T-cell immunity by mass spectrometry flow cytometry. 

Methods: Based on diagnosis and treatment guidelines of AA and literature review, the inclusion criteria were defined and patients with AA were enrolled. All patients were treated by cyclosporine with a dose of 3-5mg/kg/d, and the efficacy was evaluated after 6 months. Bone marrow samples from 30 AA patients and 4 healthy controls were collected to analyze the immune characteristics of T cells by mass spectrometry flow cytometry. Bone marrow samples were also collected from 7 patients before and after treatment, and the changes of T-cell immune characteristics were analyzed by mass spectrometry flow cytometry.

Results: We established a clinical cohort of 92 initially treated NSAA patients, all of whom received cyclosporine. We also defined the criteria for classification of transfusion-dependence and transfusion-independence. Seventy-nine patients received cyclosporine for 6 months or more. Thirty-three (41.8%) of them achieved response. Clinical indicators such as peripheral blood count and biochemistry tests prior treatment were insufficient to predict the response to cyclosporine. By analyzing data from mass spectrometry flow cytometry, we found that there were significant differences in immune characteristics on T cells between AA and healthy individuals. The expression of cytotoxic T lymphocyte associate protein-4 (CTLA-4) on CD8⁺ T cell subsets, Sphingosine-1-Phosphaphate Receptor 1 (S1P₁) on naive T cells and Interleukin 9 (IL-9) in CD4⁺ T cell subsets in AA were higher than that in healthy individuals. By analyzing the immune characteristics of T cells in the cyclosporine-treated responsive group and non-responsive group, we found that the expression level of CTLA-4 in CD8⁺ T cells at diagnosis in responders was lower than that in nonresponders (16.59 ± 1.83% vs 28.67 ± 4.22%, p＜0.05)，and the level of CTLA-4 on CD8⁺ T cells in responders was significantly increased after treatment (33.94 ± 6.38 % vs 11.28 ± 2.73 %, p < 0.05). The expression levels of S1P₁ (32.18 ± 3.46% vs 21.86 ± 2.67%, p＜0.05) and IL-9 (24.52 ± 2.97% vs 40.42 ± 4.80%, p＜0.05) on naive CD4 in responsive group were lower as compared with nonresponders.

Conclusion: As compared to healthy individuals, T cells demonstrated a signature of abnormal function in AA. The expression of CTLA-4, S1P1 and IL-9 on T cells in responders were different with non-responders, and the expression of CTLA-4 in responder patients was restored after treatment. These results suggest that T cell immune checkpoint molecules, T cell migration and differentiation are expected to be a marker for predicting the efficacy of immunosuppressive therapy, and provide a novel treatment direction for patients unresponsive to cyclosporine.