Appendices

Transcription

1 University of Groningen B cell lymphoma Wu, Rui IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite from it. Please check the document version below. Document Version Publisher's PDF, also known as Version of record Publication date: 2016 Link to publication in University of Groningen/UMCG research database Citation for published version (APA): Wu, R. (2016). B cell lymphoma: Characterization of the microenvironment and the tumor cell proteome [Groningen]: Rijksuniversiteit Groningen Copyright Other than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of the author(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons). Take-down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim. Downloaded from the University of Groningen/UMCG research database (Pure): For technical reasons the number of authors shown on this cover page is limited to 10 maximum. Download date:

2 Appendices

3

4 NEDERLANDSE SAMENVATTING Nederlandse samenvatting Het B cel lymfoom is een van de meest voorkomende hematologische tumoren over de hele wereld. De tumorcellen van lymfomen zijn afkomstig van verschillende differentiatie stadia van normale B cellen. Lymfomen kunnen verdeeld worden in 2 categorieën: non-hodgkin lymfoom (NHL) en Hodgkin lymfoom (HL). De belangrijkste subtypes van NHL zijn het folliculaire lymfoom (FL) en het diffuus grootcellig B cel lymfoom (DLBCL). Binnen HL maakt het klassieke HL (chl) 95% van alle gevallen uit. Net als in andere soorten kanker spelen interacties van tumorcellen met hun omgeving een belangrijke rol in de groei, overleving en het onderdrukken en ontwijken van de immune reactie voor en door de tumorcellen in lymfomen. Communicatie tussen tumorcellen en omgeving kan worden verdeeld in direct cel-cel contact en indirect contact via factoren die uitgescheiden worden. In dit proefschrift is ons doel om de communicatie tussen immuuncellen en tumorcellen in NHL te onderzoeken en te vergelijken met de communicatie in chl. In hoofdstuk 2 hebben we naar de veranderingen in membraaneiwitten van zeven NHL cellijnen gekeken in vergelijking met een EBV getransformeerde lymfoblastoide cellijn (LCL). We hebben, in totaal, 130 eiwitspots gevonden die meer dan een factor twee verschillend waren tussen NHL en LCL cellijnen. Daarvan waren er 28 verschillend in tenminste 3 cellijnen en deze eiwitten zijn verder geïdentificeerd. De 28 eiwitten waren vooral betrokken bij de beweging van cellen, het metabolisme en de immuun reactie. Voor 3 eiwitten hebben we verdere validatie uitgevoerd. B2M liet verlaagde expressie zien, terwijl PRDX1 en PPIA beide verhoogd waren in NHL cellijnen en weefsel van NHL patiënten. We hebben een remmer van PPIA, cyclosporine A, gebruikt om te laten zien dat PPIA een rol speelt in groei van de lymfoom cellen. Het expressie niveau van PPIA bleek geassocieerd aan de remming van celgroei, en dus lijkt PPIA een oncogene functie te hebben in NHL. In hoofdstuk 3 hebben we vervolgens gekeken naar de eiwitten die door dezelfde NHL cellijnen uitgescheiden zijn met behulp van secretoom 167 P age

5 Appendices profiling. We hebben 1317 eiwitten gevonden die uitgescheiden waren door de NHL cellijnen, waarvan 13% via klassieke secretie. Het geactiveerde B cel type (ABC) DLBCL liet het hoogste percentage unieke eiwitten zien (19%) in vergelijking met FL (8%) en kiem centrum B cel type (GCB) DLBCL (10%). Vervolgens hebben we het secretoom van NHL vergeleken met dat van HL. We vonden dat 7% van alle uitgescheiden eiwitten voorkwam in NHL en HL beide, daarnaast was het secretoom van chl het meest verschillend van de andere types met een percentage van 24% unieke eiwitten, met ABC DLBCL op de tweede plaats (18%). Aangezien het secretoom prolife alleen aangeeft dat eiwitten aanwezig zijn en geen indicatie van de hoogte van secretie niveau, hebben we ook naar de expressie op mrna niveau gekeken. Die resultaten hebben we vergeleken met de expressie van kiemcentrum B cellen (GC-B). Er waren 302 uitgescheiden eiwitten die tenminste een factor 2 verschil lieten zien in vergelijken met GC-B en 63% van die eiwitten was verhoogd in NHL cellijnen. Van de eiwitten die verhoogd tot expressie kwamen spelen de meeste een rol in de regulatie van metabolisme. Er waren 9 eiwitten met een functie in de immuun reactie. Daarvan waren cathepsine C (CTSC) en macrofaag migratie inhibitie factor (MIF) ook gevonden in het HL secretoom. Een ander eiwit, met een rol in de immuun reactie dat verhoogd was, was CD70. Deze eiwitten hebben mogelijk een rol in het ontwijken van de immuun reactie door de tumor cellen in NHL. In de volgende 3 hoofdstukken hebben we met behulp van flowcytometry naar de compositie van de omgevingscellen gekeken. Om te bepalen welke normale controle we het beste konden gebruiken hebben we in hoofdstuk 4 eerst tonsillen en reactieve lymfeklieren (RLN) vergeleken. Er waren in de hoofdgroepen meer cytotoxische T cellen (CTLs) en natural killer (NK) cellen in RLN dan in tonsil. In de subpopulaties van cellen waren er minder vroeg geactiveerde (CD69+) T cellen, CD69+ T helper (Th) cellen en CD69+ CTLs, minder GITR+ regulatory T cellen (Tregs), Th2 cellen en geactiveerde NK cellen in RLN vergeleken met tonsil. In tonsillen vonden we minder CD152+ Th cellen, CD127laag Tregs en minder NKT cellen in vergelijking met RLN. Dus de compositie van de RLN is niet hetzelfde als de tonsil. We hebben 168 P age

6 Nederlandse samenvatting vervolgens gekeken naar gesorteerde Th cellen en CTLs en genexpressie hoeveelheden vergeleken tussen tonsil en RLN, maar vonden daar geen verschillen. We kunnen concluderen dat er verschillen zijn in de immuun cel populaties tussen RLN en tonsil en dat de keuze voor het gebruik als controle weefsel goed doordacht moet gebeuren. Omdat de NHL en HL gevallen die we gebruikt hebben vooral in lymfeklieren gevonden zijn hebben we beide vergelijkingen ten opzichte van RLN gedaan. We hebben eerst de compositie van de omgevingscellen in verschillende NHLs vergeleken met RLN en met elkaar in hoofdstuk 5. Er waren minder NK cellen, macrofagen en terminaal gedifferentieerde effector memory Th cellen en meer TIA1+ CTLs in NHL vergeleken met RLN. In FL vonden we meer CD69+ T cellen, CD69+ CTLs en T follicular helper cellen en minder CTLs dan in RLN, terwijl in DLBCL er meer CD69+ CTLs en Granzyme B+ CTLs waren en minder CD127laag Tregs dan in RLN. We hebben ook de 2 subtypes DLBCL met elkaar vergeleken GCB en ABC. In GCB DLBCL waren er minder CD57+ Th cellen, TIA1+ Th cellen en CXCR4+ CTLs en meer CD127laag Tregs dan in ABC DLBCL. We hebben vervolgens het gen expressie patroon van gesorteerde Th cellen en CTLs van de NHL vergeleken met die van RLN, met en zonder activatie. Om te kijken of deze cellen geactiveerd konden worden hebben we eerst geactiveerde cellen met rustende cellen vergeleken. Aangezien een groot aantal genen significant meer dan 2 maal veranderd was kunnen we concluderen dat deze cellen niet anergisch zijn. We hebben verder gekeken naar de verschillen van NHL met RLN en vonden maar een klein aantal genen die differentieel tot expressie kwamen, dus zijn er maar weinig verschillen tussen de Th cellen en CTLs in NHL en RLN. Een van de genen die differentieel tot expressie kwam was TIGIT, een marker voor de uitputting van T cellen. TIGIT was verhoogd in NHL vergeleken met RLN in CTLs. Dit betekent dat de CTLs, alhoewel er meer zijn in NHL, mogelijk hyporeactief zijn waarschijnlijk als gevolg van uitputting. Daarnaast hebben we in hoofdstuk 6 naar de omgevingscellen van chl gekeken in vergelijking met RLN en hebben we naar het effect van een EBV infectie op de compositie van de omgevingscellen in chl gekeken. Er waren meer GITR+ en FoxP3+ Tregs in chl, in EBV+ en EBV-, 169 P age

7 Appendices vergeleken met RLN. Het percentage van CD56+CD16+ NK cellen was lager in EBV- chl dan in EBV+ chl en RLN, terwijl in EBV+ chl er meer CD69+ CTLs, Granzyme B+ CTLs, en TIA-1+ CTLs waren. Dit geeft aan dat door de EBV infectie in chl het aantal NK cellen en CTLs verhoogd waren, dit zou kunnen komen door een verhoogde Th1 immuun reactie. We zagen echter, in plaats van een verhoging in Th1 een verhoging van het aantal Tregs in EBV+ chl. We vroegen ons af wat de locatie van de verschillende cel subpopulaties was en hebben met behulp van CD26 expressie vervolgens gekeken welke cellen rondom de tumor cellen liggen (CD26-) en welke verder weg (CD26+). De CD69+ Th cellen en de FoxP3+ Th cellen bevonden zich in de tumor cel gebieden terwijl de CD25+ CTLs zich vooral buiten de tumor cel gebieden bevonden. We zagen geen effect van de EBV status op de locatie van de cellen. We kunnen concluderen dat de immuunsuppressieve cellen zich rondom de tumor cellen bevinden terwijl de immuun actieve CTLs juist buiten de tumorgebieden liggen. In conclusie, we vonden verschillen in de compositie van de infiltrerende immuun cellen in de omgeving van NHL en chl vergeleken met RLN. Verschillende eiwitten die we differentieel gevonden hebben zoals CD70, MIF, PPIA en TIGIT zijn interessante doelen voor verder onderzoek. Het mechanisme van immune ontsnapping dat de tumor cellen gebruiken is gedeeltelijk te verklaren door de hypo reactieve status van de immuun cellen en gedeeltelijk door de locatie. Behandeling met anti-tigit antilichamen of mogelijk het gebruik van PD-1 inhibitie kan mogelijk de immuun reactie tegen de tumor weer opwekken. 170 P age

8 Chinese summary 中文摘要 B 细胞淋巴瘤是一种在全世界范围内最常见的血液肿瘤 它们的肿瘤细胞具备不同分化阶段正常 B 细胞的特质 它可以被划分为两大类 : 霍奇金淋巴瘤和非霍奇金淋巴瘤 非霍奇金淋巴瘤又主要由滤泡淋巴瘤和弥漫大 B 细胞淋巴瘤组成 而在霍奇金淋巴瘤中,95% 由经典型组成 肿瘤微环境与恶性细胞之间的相互作用对于这些淋巴瘤的免疫逃逸形成产生至关重要 而这种相互作用可分为细胞与细胞直接接触或细胞之间通过分泌蛋白而间接接触 在本论文中, 我们的研究目的是调查非霍奇金淋巴瘤的免疫细胞和肿瘤细 胞之间的直接和间接的相互作用, 并进一步与霍奇金淋巴瘤中的这种相互 作用来进行比较 在第二章中, 我们研究了七种非霍奇金淋巴瘤细胞系的膜蛋白相较于 EB 病毒转化淋巴母细胞系 (LCL) 的膜蛋白变化 我们一共观察到有 130 蛋白点的表达水平在非霍奇金淋巴瘤细胞系和 LCL 细胞系相比有至少两倍及两倍以上的变化 此外, 其中有 28 蛋白与 LCL 细胞系相比, 至少三个非霍奇金细胞系的表达水平持续性改变 我们将其送做液相质谱仪器做进一步检测 这 28 个蛋白参与不同的细胞功能的调节过程, 它们主要参与调节细胞活动力, 细胞代谢以及免疫应答过程 我们观察到在非霍奇金淋巴瘤细胞系以及病人组织中人 β2 微球蛋白 (B2M) 的表达, 过氧化物还原酶 1(PRDX1) 和亲环蛋白 (PPIA) 的上调 除此之外,PPIA 的抑制剂, 环孢菌素 A 可以抑制的非霍奇金淋巴瘤细胞系的生长, 相反其对于 LCL 细胞系的生长仅有轻微的抑制作用 并且环孢素 A 诱导的细胞系生长抑制程度与亲环蛋白在该细胞系的表达水平之间存在正相关 这表明亲环素将来有潜力被用作疾病相关的生物标志物 综上所述, 我们对于非霍奇金淋巴 171 P age

9 Appendices 瘤细胞系的细胞与细胞的直接接触的相互作用的了解, 使我们进一步好奇 细胞的间接相互作用中的变化 在第三章中, 我们进一步通过分析非霍奇金淋巴瘤细胞株分泌蛋白组研究了恶性细胞和其浸润的微环境中的其他细胞之间非直接的相互作用 我们在收集的非霍奇金细胞系的培养上清液中发现了一共有 1,317 个蛋白质通过非霍奇金细胞系自身分泌的 其中 13% 的蛋白是通过经典分泌途径分泌的 与滤泡淋巴瘤 (8%) 和生发中心 B 细胞型弥漫大 B 细胞淋巴瘤 (10%) 相比, 活化 B 细胞型弥漫大 B 细胞淋巴瘤所分泌出的特有的蛋白质 (19%) 的比例最高 接下来, 我们进一步地比较了非霍奇金淋巴瘤与霍奇金淋巴瘤细胞系的分泌蛋白组学的异同 我们发现有 7% 的蛋白它们均有分泌 除此之外, 我们还观察到经典型霍奇金淋巴瘤细胞系与其他淋巴瘤细胞系差异性最大,24% 的蛋白是它们特有分泌出来的 继经典型霍奇金淋巴瘤之后, 第二个最独特的细胞系是活化 B 细胞性弥漫大 B 细胞淋巴瘤 (18%) 此外, 我们通过与用生发中心 B 细胞 (GC-B) 的比较, 分析了这些蛋白质在淋巴瘤细胞系上分泌的表达水平的变化 与生发中心 B 细胞的相比, 在非霍奇金淋巴瘤细胞系中总共有 302 个分泌的蛋白的表达水平具有至少 2 倍以上的变化 在这 302 个蛋白中的,63% 的蛋白在非霍奇金淋巴瘤细胞系中分泌增加 这些分泌增多的蛋白, 其中大多数参与调节细胞代谢 在分泌增加的蛋白质中有 9 个参与免疫应答 我们组之前已在霍奇金细胞系分泌蛋白组分析中发现组织蛋白酶 C(CTSC) 和巨噬细胞迁移抑制因子 (MIF) 的异常表达 除此之外, 非霍奇金淋巴瘤细胞系也增多了 CD70 的分泌 他们可能在非霍奇金淋巴瘤的恶性细胞的免疫逃逸中发挥了重要的作用 结合我们两部分的肿瘤细胞蛋白质组的研究, 我们发现其共同特征是调节细胞代谢相关蛋白质表达上调 肿瘤细胞蛋白 172 P age

10 Chinese summary 质的变化还能对它们所在的淋巴瘤的微环境产生影响, 因此, 我们还研究 了非霍奇金和霍奇金淋巴瘤的肿瘤微环境的组成和功能的变化 根据文献报道, 研究人员通常会选择了扁桃体或反应性淋巴结其中一者或者两者作为研究中正常淋巴组织 然而, 它们之间是否有差异或何者更适合于作为正常对照组却尚未被研究过 因此, 在第四章中, 我们见通过比较它们的细胞组成来确定何者为更适用的实验研究正常对照组 我们发现在主要细胞群中, 细胞毒性 T 细胞的和自然杀伤细胞在反应性淋巴节中的比例较扁桃体更多 在 T 细胞亚群中, 在反应性淋巴结中早期活化的 T 细胞,T 辅助细胞及细胞毒性 T 细胞,GITR 阳性调节性 T 细胞,Th2 辅助 T 细胞和活化的自然杀伤细胞的比例比扁桃体少 而扁桃体相较于反应性淋巴结具有更少的 CD152 阳性 T 辅助细胞,CD127lowT 辅助细胞, CD127low 调节性 T 细胞和更少的自然杀伤 T 细胞 以上这些提示反应性淋巴结和扁桃体的细胞组成并不相同 我们进一步研究扁桃体和反应性淋巴结中的 CD4+ 细胞和 CD8+ 细胞的是否有功能差异 在基因表达的水平, 它们之间没有找到任何功能性差异 因此我们可以说扁桃体和反应性淋巴结不能被认为是相同的正常淋巴器官, 在使用它们作为实验正常对照组的选择上应该更加地仔细进行 由于结构组成的相似性, 我们使用反应性淋巴结作为我们研究中的健康淋巴结对照组 我们在第 5 章中首先比较了不同种非霍奇金淋巴瘤的肿瘤微环境的细胞组成的不同及它们与反应性淋巴结的细胞组成的差异 我们发现在非霍奇金淋巴瘤中相较于反应性淋巴结, 有较少的自然杀伤细胞, 巨噬细胞和终末分化的效应记忆性 T 细胞和较多 TIA-1 阳性 CD8 细胞 滤泡淋巴瘤比反应性淋巴结有更多的早期活化性 T 细胞, 早期活化性细胞毒性 T 细胞的和滤泡辅助 T 细胞和较少的 CD8+ 细胞 弥漫大 B 细胞淋巴瘤中相较于反应性淋巴结, 它们有更多早期活化性细胞毒性 T 细胞和 Granzyme B 阳性 173 Page

11 Appendices CD8 细胞和少 CD127low 调节性 T 细胞 我们还研究了在两种不同亚型的弥漫大 B 淋巴瘤的肿瘤微环境的组成差异, 也即生发中心 B 细胞型 (GCB-) 和活化 B 细胞型弥漫大 B 淋巴瘤 (ABC-) 的不同 生发中心 B 细胞型与活化 B 细胞型相比有较少的 CD57 阳性 CD4 细胞,TIA-1 阳性 CD4 细胞, 和 CXCR4 阳性 CD8 细胞和较多的 CD127low 调节性 T 细胞 这些表明在不同的非霍奇金淋巴瘤中免疫细胞的组成比例不同 这些免疫细胞具有以激发免疫反应和消除恶性细胞的潜在能力 然而, 淋巴瘤尚且能形成 它使我们推测这些细胞处于低反应性的状态 因此, 我们首先考察在额外的刺激下这些免疫细胞的表现是否会有所不同 我们发现在刺激后大量基因的表达水平发生惊人地改变 这表明这些免疫细胞并非处于无能的状态 因此, 我们比较这些细胞 CD4 和 CD8 细胞在非霍奇金淋巴瘤和反应性淋巴结中的功能性差异 在在非霍奇金淋巴瘤和反应性淋巴结的 CD4 和 CD8 细胞中我们仅发现数量较少差异表达的基因 但是, 在这些差异表达的基因,TIGIT,T 细胞耗竭的一个的指标, 在所有的非霍奇金淋巴瘤的 CD8+ 细胞与反应性淋巴结相比均增加 这些观察结果表明非霍奇金淋巴瘤的肿瘤细胞的存在影响了不仅仅改变了其微环境细胞的组成, 并且还诱发了低免疫应答状态, 而该低反应状态可能是由 T 细胞耗竭导致 除了非霍奇金淋巴瘤之外, 在第六章, 我们也考察了的经典型霍奇金淋巴瘤的肿瘤微环境细胞组成的改变, 除此之外我们还研究了不同 EB 病毒感染状态对于细胞组成变化的影响 我们发现在经典型霍奇金淋巴瘤与比反应性淋巴结相比, 无论是 EB 病毒阳性或 EB 病毒阴性的霍奇金淋巴瘤, 都有更多的 GITR 阳性 Treg 细胞和 Foxp3 阳性 Treg 细胞 此外,CD56 阳性 CD16 阳性自然杀伤细胞的百分比在 EB 病毒阴性的经典型霍奇金淋巴瘤中也比在 EB 病毒阳性的霍奇金淋巴瘤和反应性淋巴结中都低 而在 EB 病毒阳性的经典霍奇金淋巴瘤中, 含有更多的早期活化的细胞毒性 T 174 P age

12 Chinese summary 细胞以及 Granzyme B 阳性 CD8 和 TIA-1 阳性 CD8 细胞 这表明经典型霍奇金淋巴瘤中的 EB 病毒感染能上调自然杀伤细胞和细胞毒性 T 细胞在其肿瘤微环境中的比例 这些可能由于由 Th1 细胞的抗肿瘤免疫应答的调节所引起 然而, 我们并没有观察到的经典型霍奇金淋巴瘤的微环境中 Th1 细胞比例增加, 与此相反,EB 病毒阳性的霍奇金淋巴瘤的调节性 T 细胞比例上升 这种现象使我们怀疑这些免疫细胞所处的位置可能也能够对经典型霍奇金淋巴瘤的免疫反应的有效性产生影响 因此, 接下来, 我们通过使用 CD26 染色来确定的经典型霍奇金淋巴瘤的肿瘤区域来进一步观察这些免疫细胞的位置 引人注目的是, 我们发现了更多的早期活化的 CD4 细胞和 Foxp3 阳性 CD4 细胞位于肿瘤区域, 相反, 更多的 CD25 阳性 CD8 细胞位于肿瘤区域之外 除此之外, 我们没有看到 EBV 感染状态在这些免疫细胞的位置有任何的影响 由此我们可以推测, 免疫抑制性细胞主要位于肿瘤细胞的周围, 而具有免疫活性的细胞毒性 T 细胞因为远离肿瘤细胞引起的经典型霍奇金淋巴瘤的免疫逃逸的发生 综上所述, 我们观察到非霍奇金和经典型霍奇金淋巴瘤的肿瘤微环境中浸润免疫细胞的组成与它们在反应性淋巴结中的组成是不同的 在非霍奇金淋巴瘤和经典型霍奇金淋巴瘤中的肿瘤免疫逃逸的机制可能是由于这些免疫细胞或处于低应答状态或由于它们所在的位置 抗 TIGIT 治疗或 PD-1 抑制剂的使用可能能够触发对于 B 细胞淋巴瘤产生有效的免疫应答 175 P age

13 Appendices 176 P age

14 Abbreviations ABBREVIATIONS IN THE THESIS ABC: AICD: AlloSCT: APC : AutoSCT: B2M: CDKN2A/B: CCL: activated B cell activation-induced cell death allogenic stem cell transplantation antigen presenting cells autologous stem cell transplantation β2microglobulin cyclin-dependent kinase 2A/B chemokine ligand CCR4: chemokine receptor 4 chl: CsA: CTL: DLBCL: DSS: EBV: ECOG : EFS: FDC: FDG-PET: FLIPI: FL: GCB-L: GC-B: classical Hodgkin lymphoma Cyclosporin A cytotoxic T cell diffuse large B cell lymphoma disease-specific survival Epstein Barr virus Eastern Cooperative Oncology Group event free survival Follicle dendritic cells fluoro-2-deoxyglucose positron-emission tomography Follicular Lymphoma International Prognostic Index follicular lymphoma germinal center B cell like germinal center B cells 177 P age

15 Appendices HL: HLA: IL: IP-10: IPI: Ig: LCL: LDH: LoDLIN: LR: NOS: Hodgkin lymphoma Human Leukocyte Antigen Interleukin interferon-induced protein-10 International prognostic index immunoglobulin lymphoblastoid cell line lactate dehydrogenase longest diameter of the largest involved node Lymphocyte-Rich not otherwise classified MALDI-TOF: matrix-assisted laser desorption/ionization time-of-flight MC: MCP-1: Mixed Cellularity Monocyte chemoattractant protein-1 NCCN-IPI: the National Comprehensive Cancer Network International prognosis index NHL: NK: NLP: NLPHL: NOS: NS: PFS: non-hodgkin lymphoma natural killer cells odular lymphocyte predominant nodular lymphocyte predominant Hodgkin lymphoma not otherwise specified Nodular Sclerosis progression-free survival 178 P age

16 Abbreviations PPIA: Peptidylprolyl isomerase A R-ACVBP: rituximab, doxorubicin, vindesine, cyclophosphamide, bleomycin, and prednisolone R-CHOP: rituximab, cyclophosphamide, hydroxydaunorubicin, oncovin, prednisone R-CVP: RLN: SDF-1: rituximab, cyclophosphamide, vincristine, prednisone reactive lymph nodes stromal cell-derived factor-1 SELDI-TOF: Surface-enhanced laser desorption/ionization time-of-flight STAT6: signal transducers and activators of transcription 6 TEMRA: Tfh: TFL: Th cells: Terminal differentiated effector memory T cells T follicular helper Transformed follicular lymphoma T helper cells TIGIT: T cell immunoglobulin and immunoreceptor tyrosine-based inhibitory motif domain Tregs: regulatory T cells 179 P age

17 180 P age

18 Acknowledgement ACKNOWLEDGMENT How time flies! I am getting to the end of my PhD study, I started to recall my whole study path. On my way, I have received so many help, love, encouragement and support from my dearest teachers, friends as well as my family. Without you, I could not go this far, therefore, I really would like to express my sincere gratitude to all of you! First and foremost, I would like to thank my dearest promoters Prof. Anke van den Berg, Prof. Jen-fu Chiu, and dearest co-promoters Dr. Lydia Visser and Dr. Arjan Diepstra for their supervision and help in the research. Dear Anke, thank you for applying me this brilliant opportunity to study in our excellent research group. During those productive meetings and discussions, I am deeply impressed by your passion to science, your logic and rigorous scientific thinking, quick mind, excellent memory, well organization and high working efficiency. I learned a lot from you. Although you have a really tight schedule every day, you are still willing to spend on as much time as you could on supervision. In addition to that, your encouragement always inspired me to face all the obstacles in research. Besides the scientific field, you are like our good friend. I really enjoy talking with you. You shared with us about your personal experiences not only about science but also about life. You care about all the students and are willing to try your best to help us out when we face with difficulties. I hope that in the future we could get a chance to further collaborate as what we discussed before. Dear Professor Chiu, I would like to express my deepest gratefulness to you. It is my honor to become your student. I am moved by your deep passion and sacrifice for scientific research. During the very first lab meeting we had, your rigorous scientific thinking and your wealth of knowledge have impressed all the students in the lab. Besides that, your physical strength also beat us easily, we were far behind you when we climbing the hills behind our main campus. You taught us not only scientific knowledge, but also how to be a proper man. I learned not only scientific knowledge, but also the baking technique from you. I really 181 P age

19 Appendices appreciate your invitation to visit your home in US. Hopefully I can visit you and Lucia next time and learn how to draw a picture from her. I really love her paintings. Dear Lydia, I want to express my deepest gratitude to you. How lucky I am to have you as my daily supervisor! You opened a door of a whole new world for me: Immunology. Without you, this whole thesis and you are the one who always back me up, who always have time for me, who always help me out, who always encourage me. How can I forget all the inspirational discussions we had, how can I forget your brilliant memory and sharp mind during those discussions, how can I forget your thoughtfulness, how can I forget your big smile, how can I forget the dinners at your places, how can I forget how delicious your deserts are, how can I forget those wonderful times we had together. You are one of the nicest people I have ever met. To me you are not only my teacher, but also like my friend and my mother in Groningen. I can hardly imagine how hard I will miss you. Dear Arjan, I would like to thank you for being my co-promoter. You are so smart. Thank you for raising so many valuable suggestions during discussions. Thank you for your dedication of precious time to help me to score the slides. Thank you for all the help during our conference trip. My sincere gratitude also goes to the member of the reading committee, Prof. Annette S.H.Gouw, Prof. AnneMieke H. Boots and Prof. Megan S. Lim. Thank you all for your time and effort you spent on reading and evaluating my thesis. Dear Annette, thank you for your kind greetings and encouragement during the past several years. Dear Mieke, thank you for your comments and suggestions for my thesis. Dear Megan, it is really nice meeting you during the ASH conference. I am really appreciated your kind words and encouragement. Next, I would like to express my appreciation to Dr. Joost Kluiver. Dear Joost, thank you for helping out with GeneSpring analysis and all the valuable suggestions and inspiration questions during the discussions. They helped me to view my researches in different angles. Besides that, I impressed by your sense of humors as well. I also appreciate the help 182 P age

20 Acknowledgement from Prof. Philip Kluin. Dear Philip, thank you for your valuable comments for my conference abstracts and also helped me to define the subtypes of lymphoma tissues. Not only your passion to science but also your kindness and generous to students and other people (the donation for pediatric patients) deeply impressed me. I also would like to express my gratitude to Prof. Guanwu Li. Dear Prof. Li, you taught me not only scientific knowledge but also the truth of life. You always helped me out when I meeting with the obstacles in experiments. I really enjoy the discussions about logic thinking in science as well as other interesting topic with you. My sincere gratitude also goes to my dearest supervisor Dr. Feng Liu. I started working under the supervision of Dr. Liu from He is one of the most important people in my life, who guided me to Hematology field. Dear Dr. Liu, without your supervision, your support and your encouragement, I could not go this far. You are a vivid example for me, to teach me how to be an excellent doctor, who always thinks about patients. You care about our study, personal life, as well as future development. You always back us up and help us out. You and Shimu are just like my parents in Shantou. I really appreciate your and her kindness for the lifelong time. I also would like to express my appreciation from the bottom of my heart to Dr. Frieda Law. Dear Frieda, how can I forget that afternoon eight years ago, I first got the chance talking with you. Although you were so busy, you still made some time for me. How lucky we are to have you there in our university!! Your vigorous attitude to the clinical work gave us vivid lessons for how to become a qualified doctor, your dedication in the hospice project for the last-stage cancer patients from poverty have already help millions of patients all over our country to have better quality and higher dignity of life. Being an volunteer of it taught me a good lesson for understanding those patients better after talking and seeing their suffering. Besides these, you also always care about our personal development. You always create development opportunities for students and doctors in our university. Without you, I definitely could not go this far. 183 P age

21 Appendices Next, I would like to thank Prof. Elisabeth G.E. de Vries and Prof. Gerwin A. Huls for offering the precious opportunity of clinical training for me. It helps me to combine my research with the clinical knowledge perfectly. My highest gratitude also goes to all the people in our research group. Dear Bea, I still can recall the day that I was introduced to you by Lydia. She said you are the most important person in our group. You are, indeed! I started nearly all my experiments with you together. You are such a kind person, I feel really comfortable to work together with you. Although you are such busy, you always were always willing to help us out. Without you, I could not start my real experiments such quick. You made everything easier for me. It is so kind of you for willing to be my paranymph. Dear Rianne, I am really grateful for your company for so many years in the lab. The whole friendship started by I learning immunohistochemistry from you in the O&O. I really enjoy chatting with you about everything. I am so glad to have you there. It made my experiments time more enjoyable. I also really appreciate your taste of clothes. Thank you for being my model for the lab photo shooting. I also appreciate your kindness for being my paranymph. Good luck with your PhD study. I am expecting to read your thesis in coming years. Dear Lotte, I owe you my deep gratitude. We collaborated in the proteomic projects. Without you, I could not finish my proteomic experiment such smoothly and such quick. You are always well organized. We were impressed by the tidiness of your desk when you moved in our office. I am so glad to witness three of the most important things happened in your life: your engagement, your marriage and your pregnant. Thank you so much for the beautiful decorations on my desk for the celebration of my birthday. Wish you all the best in your internship and your research projects!! Dear Myra, thank you for your hard work on CsA inhibition experiment! Wish you all the best in your Pharmacology career. Dear Hans, thank you for your support and your help during the past several years. If it is not the limitation of number of people, I even want to invite you to become my paranymph as well. :P. You are so nice and kind. You always pay attention to what people request and need and help us out. I really appreciate that. Dear Debora, thank you for help me 184 P age

22 Acknowledgement out with the isolation of samples and qrt-pcr experiments. Your well organized system gave me deep impression. I was glad that you like my sweet and sour chicken. Maybe next time I can cook something else for you. Dear Jasper, I am grateful for your help with my experiment as well. I know you have lots of work to do every day, you still managed to help me out such quickly. I really appreciate your thoughtfulness. Dear Mina, our friendship started from the international office; at that time we did not know that we were going to be colleagues and friends for so many years. I love your Kuku and I enjoy our ice skating trip to Utrecht very much. It was my first time to skate on ice and watched an Iranian movie. Good luck with finishing your thesis. Dear Kushi, you are the second person I met in our group. You are such a nice colleague that who always are willing to help me out. I always bother you with all sorts of questions, surprisingly, nearly all the times you have solutions for me. You sometimes surprised me by your thoughtfulness. How can I forget the stupid trip I made to Zwolle, luckily it turned out to be not such terrible finally. But your patience really impressed me. Thank you for your kindness even allowing me to bother you again this time for the formatting!! Wish you good luck in finding the next post-doc position. Hopefully this time it will have a happy ending. Dear Zheng Liang, you are the first Chinese colleague I met in our group, you were so shy when we first met. You just like a big brother to me. Thank you for making my life already not such lonely from the very early beginning. Wish you and Ning sister all the best. Dear Yuxuan, you are one of the smartest and pretty girls I have ever met. Thank you for sharing all kind of information with me. I really enjoy chatting with you. I always can learn something from you. Wish you all the best in Columbia University and wish Mr. Bai graduated smoothly this year. Dear Marcel, I am really grateful for knowing you. We shared same passion for hematology. Thank you for all the suggestions you gave on my presentations and my papers. Thank you for letting me joins your outpatient practice and explaining everything to me so well. I can see you to become a really nice doctor and teacher in the future. Wish you all the best in your practice and your projects. Dear Ali, thank you for sharing Iranian culture with us. It helped me to know more about your country. Wish you success in your coming defense and seeking for future job. Dear Melanie, you are 185 P age

23 Appendices one the most talented and smart colleague I have ever had. You got talent not only in science but also in acting. I bet you might be the best actress among the scientist, and the best scientist among all the actresses. I am expecting reading your name on both scientific Journals as well as on gossip news papers. Dear Nato, we shared the room for more than one year. I really admire your fluent English. From you, I know better about Georgia. I really miss the interesting talks we had. Good luck with your new position in Columbia University. Dear Reeny, you are such a sweet girl always with a big smile on your face. I really impressed by your hard working and your positive attitude. Good luck with finishing your projects. Do not worry, you will get there really soon! Dear Yuan Ye, thank you for your company. You just like my sister. I really had good time at your place during Spring Festivals! It made me not feeling lonely during those times. Good luck with your and Weijie s projects and future defense. Dear Geok Wee, I really appreciate your help with the microarray experiments. Wish you success with your projects! Dear Benhui and Cheng Chen, thank you for bringing sample for me to here from Wuhan. I had good time playing with Qiqi. Wish you good luck in finishing writing your thesis and wish Qiqi a better health. I also would like to thank other collegues in our group, Dear Wouter, Ahmad and Emine, good luck with finishing your thesis and success in your coming defenses. Dear Agnieska, you are such a hard working person and have passion for science. Wish you all the best with your experiments and your future career. Dear Mathilde and Jan, good luck with your projects! Dear Fubiao, you are such a nice guy. I am really happy to know you and Dandan. Good luck with all your experiments. I am expecting good news from you in coming 6 months!! I also would like to thank my dear officemates: Maaike, Jennie, Karo, Hataitip, Juan. You are just like my sisters. Your encouragements and your supports mean a lot to me. How lucky I am to have you as my officemates. I would like to express my gratefulness to people who I know in our department, pathologists Prof. Hollema, Prof. van Hemel, Dr. Hylkema, Dr. Rosati, Dr. Kats; Jolanda and Rimada from cytology, thank you for your kindness, greeting, and concerns. I also would like to thank all the secretaries, Ellen, Harriet, Ingrid, Jenney, Marijke; and 186 P age

24 Acknowledgement Richard in our departments. You always helped me out when I met with troubles I also would like to express my gratitude to my Chinese friends: Rong Wang, Juan song, Xueting Bai, Cuifeng Wang, Haoxiao Zuo, Bing Han, Hao Wang, Pieter-Jan, Jun Li, Ting Zhao, Yi Jiang, Qi Wang, Huifang Yin, Jing Han, Yuan Huang, Yuan He, Ranran Li, Ee Soo Lee, Kai Zhou, Xiaoyun Zhou, Xiaomei Yan, Chao Pang, Hui Ai, Zhuoran Yin, Naishi Li, Hui Chen, Peng Wang, Rui Yan, Chengchen Ren, Yana Geng, Yang Jing, Ni Zeng. Dear Rong, you are my positive energy bank. I really enjoy hanging out, shopping and chatting with you. I really miss our regular Friday evening gathering! Wish you good luck with your future career! Dear Juan, what a smart girl, thank you for being my officemate for the past one year. You saw me all the up and downs, we supported each other. You really helped out of the design of the cover of my thesis. Good luck in finishing writing the thesis and success in your coming defense! Dear Xueting, thank you for supported me for so many years. How lucky I am to know you. Without you, I guess I would feel really panic to go abroad alone. Due to you, I feel relax and safe when I arrived Groningen. I am really glad we live together for a month together last year, which made us closer and knew each other better. I wish you all the best in finishing the adaption of your thesis and could defend yourself as soon as possible. Good luck with Yunsong s projects as well. Dear Haoxiao, I am so happy to know you and found out that we shared so many similar interests! It caused us to become friends right away after we met. You just like my younger sister. I am grateful for your willing to become my paranymph. The whole PhD study is full of ups and downs. No worries! In the end we will conquer all of them. Dear Bing, thank you for help me to bring a surprise to my family and for cooking some hometown dishes for me. Good luck with Haoxiao s projects and Bing s defense!. Dear Huifang, thank you for your company for the last one year! We had many good times together! Through you I really saw some points I did not notice before and helped me to examine myself from other angles. Dear Cuifeng, thank you for all the dinner and barbeque at your place and the travel to France. I really had a great time! Wish you all the best in Guangzhou. Dear Jun, thank you for all the traveling, all the 187 P age

25 Appendices interesting topics we discussed, all the nice comments on the food I cooked, for nice food you cooked, I wish you all the best with finishing up your PhD and hope you could find the ideal job after your graduation. Dear Hao and peter; Ting and Yi, you all are the sweetest couples I have ever met. You are so kind hearted and care about all your friends. Hope you all have a bright future! Dear Qi, you are the most optimistic girl I have ever met. I am grateful for your help and your company a lot!! Wish you all the best! Dear Hesper, thank you for all the delicious dinners and inspiration talks! Dear Peng, thank you for being so thoughtful. Good luck with your studies and your future defenses! I also would like to thank my friends from SUMC, Suping, Xiang Zeng, Junjun, Wei Dong, Yu Lei, Jiacong, Siqi, Tiantian, Mingzan, Kunliang, Dandan, Zhijun, Kui, Xiaojuan. Together with you, I feel more like home here. Wish all the best with your studies and your future work. Dear Prof. Andy Lau, thank you for your nice comments and suggestions on my projects. I really enjoy the inspirational discussion we had during your visit to RUG. Hope you and Prof. Xu all the best in your scientific career! I also want to express my gratitude of my dearest friends in CEME Groningen, Yao Zhang and Shimu, Yuchan anty, family of Shenghua, Tian Wu, Qian Zhou, Lujun, Ying Lou, Emily, Jiahui, Richard, Yiwen, David, Ginny, Jiaxi, Yuan Wu, Suxiao, Jing Jing, Yecheng, Vicky, Fiona, Xin Gui and etc. You just like my family members in Groningen. I really had great time together with you. How can I miss my dear friends, Roselina, Chloe, Nannan, Ioana, Issac, Alexys, and Николай Цонев, and Laurel, I really miss you!! The last but not the least, I would love to express my gratitude to my dearest families, especially my dear parents and grandparents for your love, your understanding, your encouragement, and your support. All that I am or ever hope to be, I owe to my family. I really want to become the proud of you. Your health and happiness are my main concern. How lucky I am to have all of you in my life! I want to express my deep sincere apologies if I miss anyone. Although I will finish my PhD here soon, I will keep in touch with you for life long time!! 188 P age

26 Biography BIOGRAPHY Rui Wu was born on October 10 th, 1985 in Wuhan, Hubei Province, China. She finished her secondary school education in 2003 and got admitted to the Medical College of Shantou University. In 2008, she received her Medical Bachelor diploma. Due to her interest in hematology, she continued her master study with both clinical and clinical related research training under the supervision of Dr. Feng Liu at the department of Hematology and Oncology, 2 nd affiliated hospital of Shantou University Medical College, China. During this research internship, she investigated the role of reactive oxygen species in Curcumin-induced apoptosis in leukemia malignant cells. In 2010, she obtained the Certificate of Medical Doctor. In 2011, she was selected as a PhD student in Lymphoma group lead by Prof. Dr. Anke van den Berg, Prof. Dr Jenfu Chiu, Dr. Lydia Visser and Dr. Arjan Diepstra at the department of Pathology and Medical Biology, University Medical Center Groningen, the Netherlands. Her main research focus was on the characterization of the microenvironment and the tumor cell proteome of B cell lymphoma. After her defense of her PhD thesis in March 2016, she intends to continue her academic journey as a hematologist and as a researcher, which can combine her interests perfectly. 189 P age

27 Notes 190 P age

28 Notes 191 P age

29 Notes 192 P age