Title Page
감사의 글
Contents
Abstract 11
I. Transduction of the tyrosine mutant form of ZAP-70 inhibits T cell activation 13
1. Introduction 13
2. Materials and methods 18
2.1. Construction of the Escherichia coli expression vector 18
2.2. Purification of recombinant protein 18
2.3. Cell preparation 19
2.4. Cell viability assay 19
2.5. Western blots 20
2.6. T cell activation 20
2.7. SEAP assay 21
2.8. ELISA for detecting the concentration of IL-2 21
2.9. Flow cytometric analysis of CD69 expression 22
3. Results 23
3.1. Transduction activity of the Hph-1-ZAP-70-Y319F mutant into 293T cells 23
3.2. The Hph-1-ZAP-70-Y319F mutant suppresses T cells activation 26
3.3. A synergic inhibitory effect of the combination with Hph-1-ZAP-70-Y319F and Hph-1-ctCTLA-4 on the T cell activation 28
4. Discussion 34
II. Delivery of a C-terminal truncated form of SLAP with the protein transduction domain HPH-1 down-regulates T cell signaling 36
1. Introduction 36
2. Materials and methods 40
2.1. Construction of the Escherichia coli expression vector 40
2.2. Purification of recombinant protein 40
2.3. Cell preparation 40
2.4. Immunoprecipitations 41
2.5. Proliferation assay 41
3. Results 42
3.1. Transduction activity of SLAP-T-mutant-Hph-1 into cells 42
3.2. SLAP-T-Hph-1 suppresses the activation of Jurkat T cells 42
3.3. SLAP-T-Hph-1 suppresses activation of primary T cells 48
4. Discussion 55
References 57
Abstract in Korean 68
Fig. 1.1. Schematic diagram showing mechanism of PTD fusion protein entry into the cell. The PTD fusion protein binds heparan sulfate or unknown receptors on the cell surface and enters either via endocytosis or macropinocytosis. The endocytic vesicles fuse... 14
Fig. 1.2. Signaling cascades stimulated by the TcR. Schematic depiction of key protein components of major signaling cascades that are stimulated following recognition of antigen by the TcR. Differential coloring identifies biochemical events and proteins with... 16
Fig. 1.3. Purification and transduction of Hph-1-ZAP-70-Y319F. (A) To purify Hph-1-ZAP-70-Y319F from the HMS174 E. coli strain, the culture was grown at 25℃ with IPTG and the lysate was loaded onto a HisTrap chelating column. The eluted protein was... 24
Fig. 1.4. Transduction of purified proteins into 293T cells. (A) Western blot analysis of the level of ZAP-70 in 293T cells 2 h after transduction of the purified proteins. Lane 1 : control; Lanes 2-5: induced cell lysates for Hph-1-ZAP-70-Y319F (50, 100, 200 and 500... 25
Fig. 1.5. Hph-1-ZAP-70-Y319F transduction results in impaired AP-1 and NFAT-mediated gene transcription. Measurement of SEAP in (A) AP-1 and (B) NFAT transfectants incubated with purified proteins (10, 50, 100, and 200 nM) and activated with... 27
Fig. 1.6. Transduction of Hph-1-ZAP-70-Y319F results in impaired IL-2 expression. The measurement of IL-2 in Jurkat T cells (A), primary CD4+ T cells (B), and CD8+ T cells (C) incubated with purified proteins (10, 50, 100, and 200 nM) and activated with plate-bound...(이미지참조) 29
Fig. 1.7. Hph-1-ZAP-70-Y319F induces decreased CD69 on T cells in a concentration-dependent manner. Jurkat T cells (A), primary CD4+ T cells (B), and primary CD8+ T cells (C) were incubated with purified protein (200 nM) for 2 h and stimulated by plate-bound...(이미지참조) 30
Fig. 1.8. Hph-1-ZAP-70-Y319F and Hph-1-ctCTLA-4 induce decreased CD69 on T cells synergistically. (A) Jurkat T cells were incubated with Hph-1-ZAP-70-Y319F and Hph-1-ctCTLA-4 and stimulated by plate-bound anti-CD3 plus anti-CD28 for 18 h.... 31
Fig. 1.9. Hph-1-ZAP-70-Y319F and Hph-1-ctCTLA-4 reduce IL-2 production by T cells synergistically. (A) Jurkat T cells were incubated with Hph-1-ZAP-70-Y319F and Hph-1-ctCTLA-4 and stimulated by plate-bound anti-CD3 plus anti-CD28 for 18 h.... 33
Fig. 2.1. Presentation of an antigen by the major histocompatibility complex (MHC) to a T cell initiates the formation of a supramolecular-activation complex (SMAC), which is also known as the immunological synapse. T-cell-receptor (TcR) activation... 38
Fig. 2.2. Purification and transduction of SLAP-T-Hph-1 protein. (A) To purify SLAP-T-Hph-1 and SLAP-T in the Star (DE3) pLysS E. coli strain, the culture was grown at 25℃ with IPTG. The lysate was loaded on a HisTrap chelating column. The eluted... 43
Fig. 2.3. Transduction of purified proteins into Jurkat T cell. (A) Western blot analysis of the level of SLAP in Jurkat T cells 2 h after transduction of purified proteins. The cells were lysed with lysis buffer, and the lysates were resolved by 12.5% SDS-PAGE and... 44
Fig. 2.4. Effect of SLAP-T-Hph-1 and SLAP-T in Jurkat T cells. Western blot analysis of ZAP-70-Tyr319, LAT-Tyrl91, and Erk phosphorylation. Jurkat T cells were treated with SLAP-T-Hph-1 and SLAP-T (2 × 105/microtube/100 ㎕). Four hours after transduction,...(이미지참조) 46
Fig. 2.5. SLAP associates with ZAP-70 and LAT upon TcR activation. (A) SLAP-T-Hph-1 and SLAP-T-treated Jurkat T cells wete activated with anti-CD3 and CD28 antibody. Anti-HA antibody was added to 200 ㎕ of cell lysate and incubated with gentle rocking... 47
Fig. 2.6. Transduction of SLAP-T-Hph-1 results in impaired AP-1 and NFAT-mediated gene transcription. Measurement of SEAP by (A) AP-1 and (B) NFAT in transfectant incubated with the indicated concentrations of purified proteins (Lanes 3-5: SLAP-T-Hph-1,... 49
Fig. 2.7. SLAP-T-Hph-1 induces decreased expression of CD69 and IL-2 production on Jurkat T cells. (A) Jurkat T cells were incubated with purified proteins (4 μM) and stimulated with plate-bound anti-CD3 plus anti-CD28 for 18 h, and CD69 expression was... 50
Fig. 2.8. Viability and proliferation of purified protein-treated mouse splenocytes. (A) Mouse splenocytes were treated with the indicated concentrations of SLAP-T-Hph-1 and SLAP-T for 72 h. Cell viability was monitored by WST-8 assay. (B) Mouse splenocytes... 52
Fig. 2.9. Inhibition of CD69 expression by SLAP-T-Hph-1 in primary T cells. Primary CD4+ or CD8+ T cells were purified from mouse splenocytes using a MACS column with CD4 or CD8 microbeads. Mouse primary CD4+ (A) or CD8+ (B) T cells were incubated...(이미지참조) 53
Fig. 2.10. Inhibition of IL-2 secretion by SLAP-T-Hph-1 in primary T cells. Primary CD4+ or CD8+ T cells were purified from mouse splenocytes using a MACS column with CD4 or CD8 microbeads. Mouse primary CD4+ (A) or CD8+ (B) T cells were incubated...(이미지참조) 54