View : 213 Download: 0

Full metadata record

DC Field Value Language
dc.description☞ 이 논문은 저자가 원문공개에 동의하지 않은 논문으로, 도서관 내에서만 열람이 가능하며, 인쇄 및 저장은 불가합니다.-
dc.description.abstractNaive T cells in lymphoid tissues respond to antigen-presenting cells (APCs) with activation and with division mediated by the formation of immunological synapses (IS). The immunological synapses allow for T cell receptor (TCR) clustering and sustain T cell signaling. Once activated, T cells are able to circulate through blood stream and trans-migrate to sites of inflammation. At these sites the activated T cells, termed effector T cells, recognize the APCs and perform their effector function. Most studies of T cell function to date have focused on the initial activation of naive T cells, and knowledge of how T cell activation signals are amplified and sustained is limited. In our laboratory, LIME (Lck-interacting membrane protein) was identified as Lck-binding transmembrane adaptor protein (TRAP). In this thesis, I suggest that LIME is an inducer of T cell signal amplification and a mediator of effector T cell activation. In part I, the role of LIME in T cell activation was explored. LIME is preferentially expressed in hematopoietic cells, and its expression is upregulated in response to TCR stimulation. LIME localizes to membrane lipid rafts via palmitoylation and is recruited to IS upon conjugation with APCs. In addition, crosslinking of T cells expressing chimeric CD8-LIME causes the recruitment of various SH2-domain-containing molecules such as Lck, PI3K, Vav, SHP2, Gads and Grb2. Crosslinking of LIME-expressing cells also results in the phosphorylation of ERK and JNK. Furthermore, overexpression of LIME leads to the induction of interleukin-2 promoter activity. Altogether, these results indicate that LIME is a positive regulator of TCR-mediated signaling and is likely to act by recruiting signaling proteins to lipid rafts. The fact that LIME is inducible suggests that it operates at late stages of T cell activation. In part II, the role of LIME in IS formation was studied. In the IS, LIME localizes in the peripheral-supramolecular activation cluster (p-SMAC), which is enriched in adhesion molecules. Moreover, LIME associates with Vav, a known regulator of actin polymerization and integrin clustering. When overexpressed in Jurkat T cells, LIME markedly increased both actin polymerization and integrin-mediated adhesion to ICAM-1 following TCR stimulation. Because IS formation is dependent on actin rearrangement and integrin clustering, I addressed whether LIME is involved in these processes. Downregulation of LIME using LIME siRNA resulted in the abrogation of IS formation. Thus, LIME appears to control IS formation and TCR-mediated cell adhesion through association with Vav. Based on these results, I propose that LIME is a positive regulator of IS formation and that it links TCR-proximal signaling to integrin clustering. After antigen clearance, most activated T cells rapidly die through a process known as activation-induced cell death (AICD). The death of activated T cells is important for T cell homeostasis and is also involved in peripheral T cell tolerance. Although the mechanisms of AICD have been extensively investigated, the details of crosstalk between TCR-mediated signaling and T cell apoptosis remain to be elucidated. I also addressed the involvement of LIME in mediating the apoptosis of activated T cells. In part III, the role of LIME in T cell homeostasis was examined in LIME-deficient mice. To verify the involvement of LIME in effector T cell death, AICD was compared in T cells from wild type (WT) and LIME-deficient mice. In both in vitro assays using anti-CD3 monoclonal antibodies and in vivo assays using the SEB superantigen, LIME-deficient T cells were significantly more resistant to AICD than were wild type T cells. The levels of many pro-apoptotic and anti-apoptotic molecules were assayed, revealing that Bim levels were decreased but FLIP levels were increased in activated T cells from LIME-deficient mice as compared to the wild type mice. Furthermore, LIME deficiency resulted in the accumulation of effector/memory T cells, indicating that LIME is an essential molecule in T cell homeostasis. In summary, this thesis provides insights into the dual roles of the inducible adapter molecule LIME in the activation and apoptosis of T cells. On one hand, LIME promotes the activation and proliferation of T cells by strengthening IS and by recruiting signaling molecules. The inducible nature of LIME distinguishes it from other adaptor molecules such as LAT (linker for activation of T cells), which plays an essential role in early T cell activation and development. On the other hand, LIME augments AICD of activated T cells at late stages of immune responses. LIME is likely to be important in the prevention of autoimmune disease through maintaining the balance of T cell homeostasis.;효과 T 세포 (effector T cell)는 naive T 세포의 활성화 과정을 통하여 분화한 세포군으로써 병원체를 제거하는 면역 반응에 직접 관여한다. 효과 T 세포는 naive T 세포와는 다른 단백질 발현 양상과 활성화 과정을 가지는 것으로 알려져 있다. 또한 효과 T 세포는 활성 후 세포사멸 (Activation-Induced Cell Death: AICD) 과정에 의하여 제거된다. AICD는 항원의 제거 이후에 세포의 증식을 종결하여 림프구의 항상성 유지에 중요한 역할을 한다. 즉, 효과 T 세포는 외부 항원을 제거하는 역할을 함과 동시에 항원의 지속적인 자극을 받음으로써 세포사멸 (apoptosis)에 의하여 죽는다. 효과 T 세포의 활성화와 세포사멸과 같은 후기 면역 반응을 매개하는 분자에 대한 연구는 미흡하다. 본 논문은 효과 T 세포에서 발현이 유도되는 transmembrane adaptor 단백질인 LIME (Lck-Interacting Membrane protein)의 기능 연구를 통하여 후기 면역 반응의 조절 기작을 규명하였다. 제 1 장에서는 LIME의 발현 양상의 특징 및 T 세포의 활성화 과정에서 LIME의 기능을 규명하였다. LIME은 전형적인 transmembrane adaptor protein (TRAP) 단백질로써 hematopoietic 세포에서 특이적으로 발현하며, T 세포 수용체 (T cell receptor: TCR)를 통한 활성화 시에 발현양이 증가한다. 또한 LIME은 항원제시세포 (Antigen-presenting cell: APC)와의 결합 시에 면역시냅스에 위치 하였다. 본인은 T 임파구 활성화 과정에서 LIME의 기능을 연구하기 위하여CD8-LIME을 과발현하는 Jurkat T 세포주 혹은 siRNA 발현을 통하여 LIME의 발현이 억제되는 T세포주를 이용하였다. TCR을 통한 세포 활성 유도시, LIME은 빠르게 인산화 되어 Lck와 결합하고, 그 외 PI3K, Grb2, Gads, Vav, SHP-2등의 SH2 영역을 보유하는 신호전달 단백질들과 결합하였다. 또한 LIME의 과발현 시 ERK1/2및 JNK 의 활성이 증가하였고, T 세포의 증식에 중요한 인터루킨-2 promoter의 활성이 높았다. 이와 같은 결과는 LIME 단백질이 세포막에 위치하면서 TCR 신호를 전달하는 signaling complex의 scaffold 역할을 수행하는 TRAP 단백질이며, T 세포의 활성화와 증식 유도에 필수적임을 시사한다. 제 2 장에서는 T 세포의 활성에 필요한 면역 시냅스 형성과 유지 과정에서 LIME의 기능을 규명하였다. LIME은 adhesion 단백질이 밀집된 p-SMAC에 위치하며 T 세포 활성화시 actin polymerization유도와 integrin clustering을 통하여 면역 시냅스의 형성을 조절한다. 연구 결과 LIME은 Vav와의 결합으로 actin polymerization 을 조절하고 ICAM-1에 대한cell adhesion을 증가시켰다. 또한 LIME은 Vav의 GEF 활성을 유도하여 Rac1의 활성과 Pyk2의 인산화를 증가시켰다. 즉, LIME은 T 세포와 APC간의 면역시냅스를 조절하여 T 세포의 활성화와 증식을 유도한다. 제 3장에서는 LIME과 활성화된 T 임파구의 세포사멸과의 관련성을 연구하였다. T 세포 활성화에 의해 발현 양이 증가하는 LIME의 특징은 효과T 임파구의 세포사멸 조절에 관여할 가능성이 있다. 이와 같은 가설을 증명하기 위하여 LIME knockout mice를 사용하여 AICD 과정을 조사하였다. AICD 기작을 위한 in vitro와 in vivo실험에서 정상 쥐에 비해 LIME 결핍 생쥐에서 세포사멸에 대한 저항성이 훨씬 높았다. 특히 LIME 결핍T 세포는 활성화 되면서 세포사멸 유도 단백질인Bim의 발현이 저해되었으며 세포사멸 억제 단백질인 FLIP의 발현이 증가하였다. 효과T 세포에서LIME에 의한 신호전달은 Bim및 FLIP의 발현 조절을 통하여 세포사멸을 유도한다. 본 논문에서는 LIME의 기능 연구를 통하여 효과 T 세포의 활성화 및 T 세포 항상성에 관여하는 새로운 기작을 분자 수준에서 규명 하였음을 밝힌다.-
dc.description.tableofcontentsAbstract = i Contents = iv List of Figures = vii List of Tables = ix Abbreviations = x 1. INTRODUCTION = 1 1) T cell receptor signaling and Lck = 1 2) Transmembrane adaptor proteins (TRAPs) = 2 3) Immunological synapses = 6 4) TCR-induced integrin clustering = 7 5) Activated T cell death and T cell homeostasis = 8 6) LIME is a novel transmembrane adaptor protein = 11 7) Research scope = 13 2. MATERIALS & METHODS = 14 1. Plasmids = 14 2. Cells and transfection = 15 3. Antibodies = 15 4. Isolation of murine tissue and primary T cells = 16 5. T cell activation = 16 6. Immunoprecipitation and western blot analysis = 16 7. Northern blot analysis = 17 8. Lipid raft fractionation = 17 9. Metabolic labeling of 293T cells = 18 10. Jurkat T cell-APC conjugate formation = 18 11. Measurement of calcium concentration = 19 12. Affinity precipitation using GST-PAK RBD = 19 13. Determination of actin polymerization = 20 14. Adhesion assay = 20 15. Mice = 21 16. FACS analysis = 21 17. Preparation of CD4+ T cells and IS formation = 22 18. RT-PCR analysis = 22 19. Apoptosis assay = 22 20. In vivo activation-induced cell death analysis = 23 21. In vitro activation-induced cell death analysis = 23 22. Determination of mitochondrial membrane depolarization = 23 23. ELISA assays = 24 3. RESULTS = 25 Part Ⅰ - The function of LIME in T cell activation = 25 Ⅰ-1. LIME is predominantly expressed in hematopoietic cells = 25 Ⅰ-2. The expression of LIME is induced by T cell activation = 25 Ⅰ-3. LIME localizes to membrane rafts and immunological synapses = 28 Ⅰ-4. LIME associates with Lck in T cells = 33 Ⅰ-5. Tyrosine-phosphorylated LIME associates with Lck = 33 Ⅰ-6. LIME recruits signaling molecules as a raft-anchored scaffold protein = 35 Ⅰ-7. LIME activates ERK and JNK = 40 Ⅰ-8. LIME siRNA reduces calcium mobilization following TCR stimulation = 40 Ⅰ-9. Generation and analysis of LIME-deficient mice = 43 Part Ⅱ - LIME regulates Vav GEF activity and IS formation = 49 Ⅱ-1. LIME translocates to the p-SMAC following APC conjugation = 49 Ⅱ-2. LIME associates and activates Vav in vivo = 52 Ⅱ-3. LIME controls actin polymerization = 55 Ⅱ-4. LIME mediates TCR stimulation-dependent phosphorylation and activation of Vav GEF = 59 Ⅱ-5. LIME regulates TCR-induced cell adhesion = 62 Ⅱ-6. LIME mediates immunological synapse formation = 65 Part Ⅲ - The function of LIME in the apoptosis of activated T cells = 71 Ⅲ-1. T cells from LIME-deficient mice are resistant to AICD in vitro = 71 Ⅲ-2. T cell s from LIME-deficient mice are resistant to AICD in vivo = 77 Ⅲ-3. Expression of Bim and FLIP is altered in LIME-deficient activated T cells = 80 Ⅲ-4. Aged LIME-deficient mice have a higher number of effector/memory T cells = 84 4. DISCUSSION = 87 Part Ⅰ-The function of LIME in T cell activation = 87 Part Ⅱ-The function of LIME in IS formation = 91 Part Ⅲ-The function of LIME in T cell apoptosis = 97 5. REFERENCES = 102 논문 개요 = 112 Acknowledgements = 114 Appendix = 117-
dc.format.extent4679669 bytes-
dc.publisher이화여자대학교 대학원-
dc.titleFunctional Characterization of Lck-Interacting Membrane Protein, LIME, in T cell Activation and Apoptosis-
dc.typeDoctoral Thesis-
dc.format.pagex, 117 p.-
dc.identifier.major대학원 분자생명과학부- 2-
Appears in Collections:
일반대학원 > 생명·약학부 > Theses_Ph.D
Files in This Item:
There are no files associated with this item.
RIS (EndNote)
XLS (Excel)


Items in DSpace are protected by copyright, with all rights reserved, unless otherwise indicated.