臺灣博碩士論文加值系統

Stevens-Johnson Syndrome (SJS) is a severe, life-threatening cutaneous adverse reaction, most often caused by medication. Growing evidence indicates that CD8+ cytotoxic T lymphocytes, which can be activated by specific antigen-MHC complex expressed on the surface of antigen-presenting cells (APC), are involved in the pathogenesis of SJS. In our previous studies, we found that SJS induced by carbamazepine (CBZ), a commonly prescribed anticonvulsant, is strongly associated with HLA-B*1502 allele. Because specific T cell receptor (TCR) recognition of drug-bound peptide-MHC complex is likely to be driven by HLA-B*1502 with CBZ-bound endogenous peptides, we hypothesized that T cell populations having restricted TCR usage that recognized the CBZ modified antigens may be selected during the course of the disease in the SJS patients. My thesis is therefore to aim at analyzing TCR usage of CBZ-specific T cells in PBMC or blister cells from CBZ-SJS patients.
To examine if CBZ-SJS patients share common T-cell receptor usage, I first enriched drug-specific T cells from PBMC and blister cells of patients by co-culturing with irradiated autologous EBV- transformed B cells and CBZ major metabolite, 10,11-epoxide CBZ. There was a biased reactivity of the T cells after co-culturing for 3 to 5 weeks, with CD8+ T cells increasing 1.8 to 7.5 times (from average 30% to average 90%) in three CBZ-SJS PBMC and to 85.43% in one CBZ-SJS blister, which were detected by FLOW cytometry. The mRNAs of these T cells were extracted by Trizol reagent and reverse-transcripted into cDNA. These cDNAs were used to detect both α-chain and β-chain TCR phenotype by PCR with 47 sequence specific primers for different TCRV gene types. The PCR result showed a selected pattern of these biased T cells that were composed of restricted T cell gene types comparing to normal control or patient before co-culturing. After cloning and sequencing the transcripts of each TCRV gene type, the gene rearrangement and the amino acid sequences of each TCR were analyzed IMGT website. The result showed that there was preferential TCRAV and TCRBV gene usage in the SJS patients. Among four CBZ-SJS patients, TRAV14 and TRBV3-1*01 are shared by three patients, while TRAV19*01 and TRBV7-8*03/D1*01/J2-7*01 and TRAV17*01 and TRBV28*01/D1*01/ J1-1*01 are shared by two patients. This finding suggests selective activation of T cells in the pathogenesis of CBZ-SJS. More samples and further functional studies are needed to demonstrate that these TCRs are indeed involved in the induction of T cell activation.

I. Abstract.... 1

II. Introduction 3

1-1 Pharmacogenetics 3
1-2 Pharmacogenetics and adverse drug reactions 3
1-3 Adverse Drug Reactions 4
1-3-1 What are adverse drug reactions? 4
1-3-2 Classification of adverse drug reactions 4
1-3-3 Immune related adverse reactions 4
1-4 Stevens-Johnson syndrome and toxic epidermal necrolysis 5
1-5 Carbamazepine (CBZ) 5
1-6 Pharmacogenetic study about carbamazepine -induced SJS 6
1-6-1 Metabolism variation of carbamazepine 6
1-6-2 Immune responses variation of Carbamazepine 6
1-7 A strong association between CBZ-SJS and HLA-B*1502 7
1-8 Pathogenesis of Stevens-Johnson syndrome involved by T cells 7
1-9 Introduction of T cell receptor (TCR) 8
1-9-1 Structure of T cell receptor 8
1-9-2 T cell receptor gene loci 9
1-9-3 Diversity of T cell receptor 9
1-10 How TCR interact with peptide-MHC complex 10
1-11 How do T cells recognize drugs? 10
1-12 Hypothesis and Aims 11

III. Materials and Methods 12

2-1 Patients samples 12
2-2 Culture media and reagents 12
2-3 Amplification of drug-specific T cells 13
2-4 Population of CD8+ T cells analysis by FLOW cytometry 13
2-5 CD3+ CD8+ T cell sorting by FLOW cytometry 14
2-6 Extraction of RNA from CD8+ drug-specific T cells 14
2-7 cDNA synthesis by reverse transcription 15
2-8 Evaluating T cell receptor gene expression by PCR 16
2-9 T cell receptor PCR product cloning and sequencing 17
2-10 T cell receptor sequences analysis 17
2-11 Cytotoxic Assay 18

IV. Results 19

3-1 Experimental design of TCR typing 19
3-2 Generation of drug specific T cells 19
3-2-1 T cells from PBMA rapidly proliferate after Dynabeads stimulation 19
3-2-2 Drug-specific T cells from PBMC rapidly proliferate after co-culturing with 10, 11-epoxide CBZ and autologous irradiated B cells. 19
3-2-2 Drug-specific T cells from blister rapidly proliferate after co-culturing with 10, 11-epoxide CBZ and autologous irradiated B cells. 20
3-3 Population of CD8+ T cells were analyzed by FLOW cytometry 20
3-3-1 Population of CD8+ T cells from PBMC of CBZ-SJS patients increased after co-culturing 21
3-3-2 Population of CD8+ T cells from blister of CBZ-SJS patients was detected after co-culturing 21
3-3-3 Population of CD8+ T cells from blister of CBZ-SJS patients was detected after co-culturing 22
3-4 cDNA synthesis for TCR analysis 22
3-5 T cell receptor expression analysis by PCR 23
3-5-1 T cell receptor expression of PMBC from normal control and CBZ-SJS patients before co-culturing 23
3-5-2 T cell receptor expression of PBMC and blister cells from CBZ-SJS patients after co-culturing 24
3-5-3 T cell receptor expression of blister cells from CBZ-SJS patients without co-culturing 24
3-5-3 T cell receptor expression of PBMC from CBZ-tolerant patient after co-culturing 25
3-6 Analysis of TCR transcripts 25
3-6-1 TCR transcripts were analyzed by cloning and sequencing 25
3-6-2 Alignment of the sequences from each TCRV gene 26
3-6-3 Analysis of TCR rearrangement and complementarity determining regions (CDR) by comparing to database 26
3-7 Cytotoxic assay of CBZ-SJS patient 27

V. Discussion 29

4-1 Why use Dynabeads CD3/CD28 T cell expander to stimulate T cell amplification? 29
4-2 Discussion of drug-specific T cells generation by co-culturing with 10,11-epoxide CBZ and autologous irradiated B cells. 29
4-3 Why the sample number of CD8+ dominant T cells are limited after co-culturing? 30
4-4 Several TCR types were not detected in all samples 31
4-5 How to improve the accuracy and efficiency of TCR analysis? 31
4-6 Polyclonal T cells in blister fluid from CBZ-SJS patients 32
4-7 No drug specific T cell was observed in CBZ-tolerant patient after co-culturing 33
4-8 Involvement of MHC class I and CD8+ T cells in SJS pathogenesis 33
4-9 Preferential usage of both TCR α and β chain genes were detected in CBZ-SJS patients. 34
4-10 Further experiments to verify these drug-specific T cells 34
4-11 Application of these TCR 35
4-12 Conclusion 36

VI. Reference 37
VII. Figures 40

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